Lung cancer

Pathogenesis of lung adenocarcinoma

2009-04-02T06:44:00.000-07:00

Several molecular changes frequently present in lung adenocarcinomas are also present in AAH lesions, and they are further evidence that AAH may represent true preneoplastic lesions (Figure 5.3).

The most important ﬁnding is the presence of KRAS(codon 12) mutations in up to 39% of AAHs,
which are also a relatively frequent alteration in lung adenocarcinomas [15,56]. Other molecular
alterations detected in AAH are overexpression of Cyclin D1 (∼70%), p53 (ranging from 10 to
58%), survivin (48%), and HER2/neu (7%) proteins overexpression [15,57,58]. Some AAH le-
sions have demonstrated LOH in chromosomes 3p (18%), 9p (p16INK4a, 13%), 9q (53%), 17q, and 17p (TP53, 6%), changes that are frequently detected in lung adenocarcinomas [59,60]. A study on lung adenocarcinoma with synchronous multiple AAHs showed frequent LOH of tuberous sclerosis complex (TSC)-associated regions (TSC1 at 9q,53%, and TSC2 at 16p, 6%), suggesting that theseare candidate loci for tumor suppressor gene in asubset of adenocarcinomas of the lung [60]. Activation of telomerase expressed by expression of
human telomerase RNA component (hTERC) and telomerase reverse transcriptase (hTERT) mRNA, has been detected in 27–78% of AAH lesions, depending in their atypia level [61]. Recently, it has been shown that loss of LKB1, a serine/threoninekinase that functions as a tumor suppressor gene, isfrequent in lung adenocarcinomas (25%) and AAH (21%) with severe cytological atypia, while it is rare in mild atypical AAH lesions (5%), suggesting that
LKB1 inactivation may play a role in the AAH progression to malignancy [62].
Several mouse models have been developed to better study various oncogenic molecular signaling pathways and the sequence of molecular events involved in the pathogenesis of peripheral lung tumors, and to test novel chemopreventive agents [63]. The KRAS oncogenic mouse model is characterized for the development of peripheral alveolar type of proliferations, including AAH, adenoma,and adenocarcinoma [63]. Using this mouse model,
several important ﬁndings that need to be further validated in human tissues have been reported.Kim et al. [64] identiﬁed the potential stem cell population (expressing Clara cells-speciﬁc protein and surfactant protein-C, termed bronchioalveolar stem cell, BASC) that maintains the bronchiolar Clara cells and alveolar cells of the distal respiratory epithelium and which could be considered the precursors of lung KRAS neoplastic lesions in
mice. Wislez et al. [50] provided evidence that the expansion of lung adenocarcinoma precursors
induced by oncogenic KRAS requires mammalian target of rapamycin (mTOR)-dependent signalingand, most importantly, that inﬂammation-related host factors, including factors derived from macrophages, play a critical role in mice adenocarcinoma progression. Recent ﬁndings reported by Collado et al. [65], suggest that KRAS oncogeneinduced senescence may help to restrict tumor progression of lung peripheral lesions in mice. They discovered that a substantial number of cells in mice premalignant alveolar type of lesions undergooncogene-induced senescence, but the cells that escape senescence by loss of oncogene-induced senescence effectors, such as p16INK4a or p53,progress to malignancy. Thus, senescence is a deﬁning feature of premalignant lung lesions, but not invasive tumors.

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2009-04-02T03:17:00.000-07:00

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Tumor suppressor genes 4

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RAS/RAF/MEK/ERK pathway
The RAS family of proto-oncogenes (HRAS, KRAS,
and NRAS) are 21-kD plasma membrane-associated
G-proteins that regulate key signal transduction
pathways involved in normal cellular differenti-
ation, proliferation, and survival [104]. Multiple
studies have shown that oncogenic KRAS (e.g.,
KRASV12 mutant) activates cell signaling pathways
important to cellular transformation [105]. As a
result, KRAS abnormalities represent an impor-
tant therapeutic target. RAS mutations (nearly al-
ways KRAS mutations in lung cancer) are found in
15–20% of NSCLCs, especially in adenocarcinomas
(20–30%), but never in SCLCs [26]. The mutations
occur in codons 12, 13, and 61, all of which in-
ﬂuence intrinsic GTPase activity [104]. A number
of drugs that target different aspects of RAS func-
tion and metabolism have been developed and are
currently under clinical investigation [104]. These
include the farnesyl transferase inhibitors tipifarnib
and lonafarnib, which are now being tested in thecombination with cytotoxic drugs in phase III clini-
cal trials [106].
BRAF protein serine/threonine kinase is a down-
stream effecter of the Ras pathway and mutations
of BRAF occur in ∼70%melanoma, but in only 3%
of lung cancers [107–109]. However, for those rare
lung cancers, mutated BRAF protein is a potentially
important and speciﬁc therapeutic target. An orally
administered Raf kinase inhibitor, BAY 43-9006 (so-
rafenib), is currently being tested in phase I and
phase II trials in lung cancer [110–111].
Activated BRAF phosphorylates and activates
MEK1 and MEK2, which in turn phosphorylate
and activate ERK1 and ERK2. However, MEK or
ERK gene ampliﬁcation ormutations have not been
found in lung cancers. Nevertheless, ERK1/ERK2
are constitutively activated in a subset of lung can-
cers and MEK and ERK remain therapeutic tar-
gets for lung cancer treatment using an oral MEK
inhibitor CI-1040 and its derivative PD03255901
[112].

Tumor suppressor genes 3

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Receptor tyrosine kinases
The EGFR family
The EGFR family of receptors are transmembrane
TK receptors and are composed of EGFR, HER2,
HER3, and HER4 and each has unique proper-
ties. For example, HER2 lacks a functional ligand-
binding domain and HER3 lacks kinase activity [63].
Upon ligand binding, these EGFR family members
form active homo- and hetero-dimers, leading to
autophosphorylation and activation of intracellular
signaling cascades. EGFR is overexpressed in ∼70%
ofNSCLCs but rarely expressed in SCLCs [64]. There
are several drugs targeting EGFR or HER2 currently
available including the small molecule TKIs, geﬁ-
tinib, erlotinib, and the monoclonal antibodies, ce-
tuximab (targeting EGFR), and trastuzumab (target-
ing HER2).
Recently, several mutations in the TK domain of
EGFR have been described, and are not infrequent in
NSCLC (10–20%), but never occur in SCLC [65,66].
Of interest is that TK domain mutations are almost
exclusive to lung cancer, whereas intracellular re-
gion mutations are found in glioblastomas. In lung
cancer, these mutations are limited to the ﬁrst four
exons of the TK domain and are categorized into
three different types (deletions, insertions, and mis-
sense point mutations). Inframe deletions in exon19 (44% of all mutations) and missense mutations
in exon 21 (41% of all mutations) are the most fre-
quent, accounting for more than 80% of all muta-
tions [67]. Importantly, the presence of mutations
in TK domain correlates with the drug sensitivity to
TKIs [65,66]. An intriguing characteristic of EGFR
mutations is that they occur in a highly selected
subpopulation: female East-Asian never smokers
with adenocarcinoma histology [68]. Notably, be-
fore EGFR mutations were discovered, all of same
clinicopathological factors were found to be associ-
ated with tumor responses to TKIs [69,70].
Although several studies have conﬁrmed the re-
lationship between the presence of mutant EGFR
and the response to TKIs [65,66,71], a subset of
NSCLC patients with mutant EGFRs do not respond
to TKIs. These tumors often (>50%) have a “sec-
ond” TK domain mutation (T790M) usually found
in patientswho relapse after TKI treatment, suggest-
ing its contribution to acquired resistance to TKIs
[72,73]. However, several examples of the T790M
mutations occur in lung tumors not treated with
EGFR TKIs, and often themutation is only in a small
subset of the tumor cells. This contrasts with the
other EGFR TK domain mutations, which are in all
tumor cells.Also, a germline EGFR T790Mmutation
was reported to be associated with familial NSCLC,
suggesting that this mutation could predispose peo-
ple to lung cancer [74]. Fortunately, there are EGFR
TKIs that inhibit EGFR with the T790M mutation,
and these drugs are currently under clinical evalu-
ation [75].
Some patients without EGFR mutation also re-
spond to TKIs, and several predictive markers other
than EGFR mutation have been reported to corre-
late with TKI response, including EGFR ampliﬁca-
tion, elevated EGFR protein, HER2 ampliﬁcation,
HER3 ampliﬁcation, and activation of AKT [76–
80]. In fact, KRAS mutations and EGFR mutations
are mutually exclusive. KRAS mutations are asso-
ciated with cigarette smoking, while EGFR muta-
tions generally occur in never smokers [81]. These
studies suggest that other biological features be-
sides EGFRmutation status determine TKI response.
Among biologic predictors, EGFR mutation and
ampliﬁcation by ﬂuorescence in situ hybridization
are highly correlated with TKI response whileEGFR protein expression gives conﬂicting results
[65,66,71,76,82]. There is also the possibility that
tumors with EGFR mutations are associated with
better survival independent of TKI treatment. Thus,
all survival studies after TKI treatment need to have
molecular analyses for comparison [80,83,84]. Two
well-controlled phase III studies were conducted for
these drugs. The results of these studies showed that
erlotinib prolonged survival of previously treated
NSCLC patients by 2 months (BR21 trial), while
geﬁtinib failed to show survival beneﬁt (Iressa Sur-
vival Evalulation in Lung Cancer (ISEL)) [86,87].
Despite positive preclinical studies of the combi-
nation of TKI and chemotherapy, several phase
III studies have failed to show a survival bene-
ﬁt of adding erlotinib or geﬁtinib to conventional
chemotherapy [88,89]. Finally, lung cancers with
EGFR mutations are more sensitive to ionizing ra-
diation than those without EGFR mutations, which
potentially provides a molecular basis for combined
modality treatment involving TKIs and radiother-
apy [90].
While standard criteria for selecting patients with
NSCLC for TKI therapy are being developed, in prac-
tice, East-Asian female patients with tumors that
have EGFR mutations or EGFR ampliﬁcation and
that are never smokers often receive TKI therapy. To
address this issue, prospective clinical trials designed
to incorporate the patient’s clinicopathological data
as well as molecular biological features (EGFR mu-
tation and/or ampliﬁcation) of the tumors are cur-
rently underway.
HER2 mutations occur in 2% of NSCLCs. All re-
ported HER2 mutations are in-frame insertions in
exon 20 and target the corresponding TK domain
region as in EGFR insertion mutations and occur in
the same subpopulation as those with EGFR muta-
tions (adenocarcinoma, never smoker, East Asian,
and woman) [68,91,92]. So far no small molecule
inhibitors show similar potency against HER2muta-
tions as seenwith EGFR TKIs and studies are needed
to see if mutant HER2 lung cancers respond to the
anti-HER2 antibody trastuzumab. HER4 mutations
were found in (2.3%) NSCLC tumor samples from
Asian patients including male smokers [93].
EGFR mutations occur as preneoplastic lesions
occurring in histologically normal bronchial epithe-lial cells adjacent to tumors with EGFR mutations.
The discovery of EGFR mutations could be used
as an early detection marker and chemoprevention
target [94]. Transgenic mice with either EGFR point
mutations or deletion mutations develop lung ade-
nocarcinomas with similar histology to those seen
in patients [95,96]. When the mutant gene was
“turned-off” in the mice through controlled gene
expression the lung tumors all regressed indicating
thatmutant EGFR is required for both initiation and
maintenance of the tumors.
c-KIT
SCLC but not NSCLC frequently express (40–70%)
both the receptor c-KIT and its ligand, stem cell fac-
tor (SCF) suggesting an autocrine loopmay promote
the growth of the SCLC cells [97]. However, unlike
gastrointestinal stromal tumors which frequently
contain c-KITmutations, activating c-KITmutations
are very rare in lung cancer [98,99].While imatinib,
an inhibitor of c-KIT kinase, inhibits cell growth in
some c-KIT expressing SCLC cell lines in vitro, two
phase II clinical studies and amouse xenograft study
failed to showtumor regression in SCLC by imatinib
monotherapy [100–103].

Tumor suppressor genes 2

2009-03-30T15:13:00.000-07:00

3p tumor suppressor genes
Allele loss in 3p, including LOH and homozygous
deletion, occurs in nearly 100%of SCLCs and more
than 90% of NSCLCs and is one of the earliest
events in lung cancer development. Because of the
early changes in chromosome region 3p21.3 (oc-
curring in histologically normal lung epithelium)
the presence of 3p allele loss and inactivation of
expression of these 3p TSGs can be of use in de-
termining smoking related ﬁeld effects. Three dis-
creet regions of 3p loss have been identiﬁed by
allelotyping in lung cancers, including, a 600-kb
segment in 3p21.3, the 3p14.2 (FHIT/FRAB3), and
the 3p12 (ROBO1/DUTT1) regions. The 3p21.3 re-
gion has been analyzed most extensively and 25
genes were identiﬁed from this region.
One of the best studied genes in this region is
RASSF1A, which is rarely mutated in lung cancer
but whose expression is frequently lost by tumor
acquired promoter methylation [51,52]. RASSF1A
is involved in multiple pathways critical to can-
cer pathogenesis, including cell cycle, apoptosis,
and microtubule stability. RASSF1A is methylated
in ∼90% of SCLCs and ∼40% of NSCLCs and has
the ability to suppress the growth of lung cancer
cell lines in tissue culture and as xenografts in nude
mice [51,52].
FUS1 is located next to RASSF1A and one of the
two alleles of the gene is often lost in lung cancers.
FUS1 is rarelymutated in lung cancers, does not un-
dergo promoter hypermethylation, yet the protein
product of this gene is frequently lost in lung can-
cer compared to normal lung tissues [53].Wild-type
FUS1 but not tumor-acquiredmutant FUS1 induces
G1 growth arrest and apoptosis [53].Administration
of FUS1 with in DOTAP:cholesterol (DOTAP:Chol)
nanoparticles (FUS1-nanoperticles) inhibits cancercell growth in vitro and in vivo. These preclinical
studies provide a basis for FUS1 gene therapy clin-
ical trials for the treatment of lung tumors using
FUS1-nanoparticles [54,55].
Two other 3p21.3 candidate tumor suppressor
genes, Semaphorin 3B (SEMA3B) and a family
member SEMA3F, are extracellular secreted mem-
bers of the semaphorin family, and are impor-
tant in axonal guidance. Wild-type SEMA3B, but
not missense mutant SEMA3B, induces apopto-
sis when re-expressed in lung cancers or added
as a soluble molecule [56,57]. Overexpression of
SEMA3F in tissue culture results in inhibition of
tumor cell growth and tumor cell invasion. Both
SEMA3B and SEMA3F are soluble, secreted pro-
teins, and therefore are promising candidates for
drug development.
Two other 3p genes with evidence to support
their candidacy as tumor suppressors are FHIT and
retinoic acid receptor beta (RARβ). FHIT is located
in 3p14.2, one of the most common fragile sites of
the human genome. FHIT is either homozygously
deleted or expresses aberrant transcripts in more
than 50% of lung cancers [58]. In addition, FHIT
overexpression induces apoptosis in lung cancer
cells. RARβ is located at 3p24 and functions as a
receptor for retinoic acid (RA). Although the RARβ
gene is not mutated in lung cancer, it undergoes
methylation in 72% of SCLCs and 41% of NSCLCs,
leading to loss of its expression [59]. Re-expression
of RARβ in lung cancer cell lines suppresses their
growth in the culture and nude mice [60].
Oncogenes and the pathways
they regulate
While there are multiple components to each of the
growth signaling pathways involved in lung can-
cer, we will focus the discussion on those proteins
that are frequently affected by genetic abnormalities
in cancer. It has become clear that these mutated
proteins, while driving cells toward transformation,
also “addict” the cells to their abnormal function.
This concept is referred to as “oncogene addiction”
and represents a cellular physiologic statewhere thecontinued presence of the abnormal function,while
oncogenic, also becomes required for the tumor to
survive [61]. This means that if the function is re-
moved or inhibited, for example, by a targeted drug,
the tumor cells die. By contrast, bystander normal
cells, which are not “addicted” to the mutant pro-
tein, are much less sensitive to the drug; thus, the
targeted drugs have great tumor cell speciﬁcity. The
most important example of this concept for lung
cancer is EGFR. Tumors withmutations in EGFR are
dependent on survival signals transduced by mu-
tant EGFR, and thus are particularly sensitive to ty-
rosine kinase inhibitors (TKIs) [62]. These ﬁndings
have led to massive genome-wide sequencing ef-
forts (discussed above) targeting thousands of genes
to ﬁnd additional mutated oncogene targets for ra-
tional therapeutics design.

Tumor suppressor genes

2009-03-30T15:12:00.002-07:00

Several key tumor-suppressor pathways are fre-
quently inactivated in lung cancer. These include
the p53 and the p16INK4a
—CyclinD1-CDK4-RB
pathways.
The p53 pathway
The tumor suppressor gene p53 is the most fre-
quently mutated gene in human cancer, and p53
is inactivated by mutation in ∼90% of SCLCs and
∼50% of NSCLCs, respectively [26,46]. Most in-
activating mutations in p53 are caused by point
mutations in the DNA-binding domain (missense
mutation, 70–80%) of one parental allele and LOH
(deletion) of the other. Occasionally homozygous
deletions are observed. p53 is located at chromo-
some 17p13.1, and codes for a protein that functions
as a key transcription factor. The transcriptional tar-
gets of p53 include a number of cell cycle regula-
tory proteins such as p21 andMYC, as well as many
proteins involved in apoptosis such as BAX, 14-3-
3σ, and GADD45. p53 regulation occurs primarily atthe level of protein stability. p53 controls transcrip-
tion of MDM2, an E3 ubiquitin ligase, which in turn
regulates p53 stability in a feedback loop. This par-
ticular connection in the p53 pathway is a frequent
target of dysregulation in tumor cells.
The p53 pathway is activated in response cel-
lular stress and DNA damage induced by gamma-
irradiation, ultraviolet light, DNA damaging drugs,
and carcinogens. p53 stabilization results in the
expression of downstream genes, which induces
either cell cycle arrest to permit DNA repair, or
programmed cell death when there is too much
damage. Loss of p53 function allows cells to di-
vide in spite of genetic damage, which can result
the clonal expansion of premalignant cells. In most
cases, only mutant, missense p53 is present because
of LOH involving thewild-type p53 allele. However,
in some cases, mutant p53 proteins can form het-
erodimers with wild-type p53 inactivating its tumor
suppressive function even before LOH. These “gain-
of-function” mutations contribute to increased tu-
morigenicity and invasiveness of several types of
cancers [26,46]. However, despite large-scale stud-
ies, it is not clear whether NSCLCs with p53 muta-
tions have impaired survival compared to lung can-
cers with only wild-type p53.
There are two important upstream regulators
in the p53 pathway: MDM2 and p14ARF. MDM2
functions as an oncogene by reducing p53 levels
through enhancing proteasome-dependent degra-
dation. Ampliﬁcations of MDM2 were reported in
∼7% (2/30) of NSCLCs, resulting in loss of p53
function [46]. p14ARF
derives from the p16 locus
with an alternatively spliced 5-exon that results in
an alternative reading frame for translation. p14 en-
codes a protein that binds toMDM2 thereby inhibit-
ing its ubiquitination activity,which leads to the sta-
bilization of p53. Immunohistochemistry analyses
of p14ARF on lung cancers have shown that p14ARF
protein expression was lost in ∼65% of SCLCs and
∼40% of NSCLCs. Thus, through p53 mutation or
changes in MDM2 or p14, the p53 pathway is inac-
tivated in the majority of all lung cancers.
Lung cancer cells are addicted to loss of p53 func-
tion. When wild-type p53 is re-expressed in lung
cancer cells with mutant or deleted p53, the tumor
cells undergo apoptosis. These ﬁndings have led to
clinical trials of p53 gene replacement therapy. The
results frompreclinical and early-stage clinical trials
of p53 gene replacement therapy using a replication
incompetent retrovirus p53 expression vector in pa-
tients with NSCLCs, show evidence of antitumor
activity and the feasibility and safety of gene ther-
apy [47]. INGN 201 (Ad5CMV-p53, AdvexinTM),
a replication-impaired p53 adenoviral vector has
been evaluated in clinical trials, and is both safe and
effective for the treatment of several different types
of cancer [48]. This treatment has been approved in
China for the treatment of primary head and neck
cancers in combination with radiation therapy and
is currently undergoing phase III trials in head and
neck cancer in the United States.
The RB pathway
The RB pathway plays a central role in G1/S
cell transition. Hypophosphorylated RB exerts its
growth suppressive effect by binding to and inhibit-
ing the E2F transcription factor, which promotes
cells through the G1/S transition. RB is phosphory-
lated by the CyclinD1/CDK4 complex. Once these
kinases phosphorylate RB, it releases E2F, resulting
in transition from G1 to S. Thus, loss of RB function
though deletion ormutation leads to loss of theG1/S
checkpoint, and is a common event in lung cancer,
particularly SCLCs (>90%), while inactivation of
RB is found in 15–30% of NSCLCs [26].
The activity of the CDK4/Cyclin D1 complex is
regulated by p16. p16 keeps RB hypophosphory-
lated (and growth suppressingmode) by preventing
CDK4 from phosphorylating RB. Thus, loss of p16
function results in loss of function of the RB path-
way. By contrast to RB, p16 is more frequently in-
activated in NSCLCs (∼70%) than in SCLCs (10%)
[26]. Inactivation of p16 is caused by LOH coupled
with deletion, intragenic mutations or promoter
hypermethylation of the remaining allele. In lung
cancer, promoter methylation is the most frequent
method of inactivation of p16.
Overexpression of either CDK4 or Cyclin D1
inhibits RB pathway function by saturating the
growth suppressive activity of p16. CDK4 is am-
pliﬁed in some cases of NSCLCs, but cyclin D1 is
overexpressed in more than 40% of NSCLCs as as-
sessed by immunohistochemistry [26,49]. Recently,overexpression of Cyclin D1 in normal-appearing
bronchial epithelial of patients with NSCLCs has
been reported to be associated with smoking and to
predict shorter survival, suggesting the possible util-
ity of Cyclin D1 as a molecular marker to identify
high-risk individuals [50]. Thus through changes in
either RB, p16, CDK4, or cyclin D1, this important
growth regulatory pathway is inactivated and dis-
rupted in the large majority of lung cancers.

Epigenetic basis of lung cancer—DNA methylation and tumor suppressor gene inactivation

2009-03-30T15:12:00.001-07:00

Lung cancers turn out to have at least as many
epigenetic alterations as genetic changes. Epige-
netic phenomena are heritable characteristics (phe-
notypes) that cannot be explained by differences
in the primary structure of DNA. In normal cells,
genomic DNA is packaged into chromatin. Chro-
matin regulates the spatial arrangement and acces-
sibility of DNA to transcription factors in the nu-
cleus. DNAmethylation is an important component
of epigenetic gene regulation in normal cells and its
dysregulation is crucial to cellular transformation
on at least two levels: genome-wide hypomethyla-
tion and gene-speciﬁc promoter hypermethylation.
Genome-wide hypomethylation affects heterochro-
matic regions of the genome, which do not ordinar-
ily code for protein. These regions were believed to
be transcriptionally inert, or “junk” DNA, but recent
evidence suggests that the transcriptional capacity
genome has been underestimated, and thus could
encode sequences important for cancer [20].
Genome-wide hypomethylation has several im-
plications in preneoplastic cells, affecting both
transcription and genetic integrity. Transcriptional
effects include loss of imprinting, re-expression
of genes involved in fetal development, and
transcriptional activation of repetitive elements
[19,30,31]. The genetic effects are indirect andinvolve larger-scale processes such as overall chro-
matin architecture, aneuploidy, and DNA replica-
tion [20,32,33].
There is overwhelming evidence that tumor-
acquired promoter hypermethylation, leading to
loss of expression of the associated gene, is a com-
mon event during themultistep pathogenesis of hu-
man lung cancer [26,34–37]. Over the past decade,
nearly 150 genes have been identiﬁed that show
tumor-speciﬁc methylation in primary tumor sam-
ples, includingmany in lung cancer (Table 4.2) [38].
Certain loci are preferentially methylated in certain
cancer types [39,40].Gene-speciﬁc promoter hyper-
methylation is an early event in tumorigenesis and
occurs in conjunction with transcriptional silencing
of the associated gene. In addition, aberrant pro-
moter hypermethylation often coincides with loss
of heterozygosity resulting in complete loss of ex-
pression and thus function of the affected locus
[16,37]. However, the molecular mechanisms that
drive tumor-acquired promoter hypermethylation
in cancer progression are not yet known [41].
DNA methylation-dependent transcriptional si-
lencing frequently affects genes that are involved
in transcriptional regulation, DNA repair, negative
regulation of the cell cycle, as well as growth reg-
ulatory signaling pathways (Table 4.2). Similar to
genetic changes, promoter hypermethylation in-
creases during tumor progression. However, in-
creasing promoter hypermethylation also occurs
with increasing age and with carcinogen exposure-
related cancers such as that of the colon and lung
[42]. In the lung, a continuumof increasingmethy-
lation fromhyperplasia through invasive carcinoma
is evident [27,28,34,43,44]. Aberrant promoter hy-
permethylation has been found in a variety of pre-
neoplastic lesions, which supports the hypothesis
that this epigenetic alteration is an early event in
carcinogenesis. This observation has resulted in sub-
stantial interest from the medical community in
that detection of methylation in sputum, blood, or
bronchial washingsmay have utility in the early de-
tection of cancer.
Some genes, such as the important TSG p53, are
never inactivated by promoter hypermethylation
because they do not have a promoter region CpG
islands. Other genes, such as the tumor suppressorgene RASSF1A, which has a prominent CpG island
are nearly always inactivated by LOH and promoter
hypermethylation in both SCLC andNSCLC. Thus, a
curious feature of aberrant promoter hypermethy-
lation is that it does not appear to affect all genes
with equal probability. An even more conspicuous
example of this phenomenon is evidenced by the
difference between p16 and RB; the protein prod-
ucts of these two genes interact directly and inac-
tivation of one or the genes (and thus this regu-
latory pathway) is nearly universal in tumors. In-
terestingly in SCLC, RB (13q14) is nearly univer-
sally inactivated, whereas in NSCLC, it is usually
p16 (9p21) that is lost. Both genes have large CpG
islands in their promoter regions, but only p16 is
methylated with signiﬁcant frequency, whereas in-
activation of RB almost always occurs through DNA
mutations. This suggests tumor-acquired promoter
hypermethylation is nonrandom, and that there is
something about certain loci that makes them par-
ticularly susceptible to aberrant methylation or to
mutation [37,45].

Preneoplasia and the early detection of lung cancer

2009-03-30T15:11:00.000-07:00

As discussed above, lung cancer results from the
accumulated effects of genetic and epigenetic al-
terations over time. Strong evidence for this posi-
tion derives from molecular genetic studies whichshow that some genetic alterations found in frank
tumors can also be identiﬁed in preneoplastic lung
cells. Using a series of microsatellite markers and
precise microdissection of cancer and lung preneo-
plastic lesions in smoking-damaged lung epithelium
as well as primary lung cancers, several groups have
shown that as cells progress histologically from hy-
perplastic epithelium through dysplasia, carcinoma
in situ, to invasive carcinomas, they acquire more
frequent and extensive genetic alterations [27,28].
The earliest genetic change that has been identiﬁed
in preneoplastic bronchial epithelial cells often in-
volves the short arm of chromosome 3. The speciﬁc
region is a 630-kb minimal homozygously deleted
portion of cytoband 3p21.3 [24]. This locus encom-
passes approximately 20 genes, including RASSF1A,
FUS1, and SEMA3B, which are discussed in the next
section.
Themost common genetic alterations and the rel-
ative timing of their appearance during lung tumori-
genesis are of particular interest because knowledge
of their occurrence can be potentially used for risk
assessment of who is themost likely to develop lung
cancer. However, these changes primarily represent
a “full defect” induced by cigarette smoking and
only rarely do sites of these changes progress to full-
ﬂedged cancer.
In exposure-related cancers such as lung cancer,
progenitor epithelial cell clones frequently undergo
epigenetic and genetic alterations that expand into
“ﬁelds” of cells, exacerbating the problem of clonal
instances of genetic damage. The presence of spe-
ciﬁc genetic changes such as a deﬁnedmutation can
be used to track clonally-related cells. In one such
study, a group of pathologists examined 10 widely
dispersed sites in the tracheobronchial tree of a pa-
tient who died of severe atherosclerosis and found
patches of cells with the identical p53 point muta-
tion in seven of these sites [29]. While there was
no evidence of cancer in any organ at autopsy, the
presence of this mutation indicated that a lung cell
with the stem-like properties existed and migrated
throughout the lung.
The combination of chronic exposure to cigarette
smoke and chromosomal instability lead to LOH
in 3p21.3 (several genes), 9p21 (p16), and 17p.13
(p53) and frequent ampliﬁcations in eight (c-Myc),which contained deﬁned tumor suppressor genes
or oncogenes. Loss of tumor suppressor gene func-
tion and activation of oncogenes contribute to the
initiation, development, and maintenance of lung
cancer by conferring six distinct properties, called
the “hallmarks of cancer” [9]. The hallmarks in-
clude self-sufﬁciency in growth signals (activation
of oncogenes), insensitivity to growth-inhibitory
signals (inactivation of TSGs), evading apoptosis,
immortalization, sustained angiogenesis, and tissue
invasion and metastases. In the following section,
we will discuss the genes involved in conferring
these “hallmarks” on lung cancer cells.

Chromosomal instability, aneuploidy, and loss of heterozygosity

2009-03-30T15:10:00.002-07:00

There are several types of genetic damage that con-
tribute to lung cancer pathogenesis: (i) changes in
chromosome number; (ii) changes in chromosome
structure; (iii) allelic alterations and loss of het-
erozygosity (LOH); and (iv) sequence alterations in
the form of point mutations or small ampliﬁcations
or deletions [17]. The ﬁrst three types of genetic
damage fall under the rubric of genomic instability
and can occur anywhere in the genome, whereas
the ﬁnal type involves mutations in protein coding
sequences. In this section, we will discuss genomic
instability in the context of chromosomal instabil-
ity, aneuploidy, and loss of heterozygosity. In the
next section, we will discuss the genes frequently
affected by mutational events in human lung can-
cer, and howknowledge of their functionwill trans-
late into novel, effective therapeutics.While we dis-
cuss genomic instability and loss or gain of gene
function in different sections, it is important to re-
alize that these factors are not mutually exclusive
and both contribute to cellular transformation in
complex and cooperative ways. The consequences
of speciﬁc alterations in DNA sequence, be they
large-scale translocations or single-pointmutations,
are rarely binary events; rather, it is the accrued ef-
fects of multiple, sequential genetic alterations over
time that gives each tumor its idiosyncratic clinical
course and outcome.
It has been argued that the term genetic insta-
bility properly refers to the rate at which genetic al-
terations occur [17]. Vogelstein and others correctly
argue that the rate of genetic change cannot be in-
ferred from the extant alterations in a given sam-
ple, but rather should be determined experimen-
tally. As a result, here we will distinguish between
the terms genomic instability and genetic instability,
and use the termgenomic instability to refer only to
the fact of alterations in chromosome number (ane-
uploidy) or gross alterations in chromosome struc-
ture through translocation, ampliﬁcation, and dele-
tion (chromosomal instability). Genomic instability
can involve LOH, particularly in the context of tu-
mor suppressor genes. In this case, one allele has a
mutation or epigenetic change inactivating one al-
lelewhile the otherwild-type allele is lost alongwith
many other genes leaving the cell with a completely
inactive tumor suppressor gene. This commonly oc-
curs in the case of the well-known TSGs p53, p16,
and RB.
LOH refers to the loss of one allele of a given lo-
cus, but says nothing about the number of copies
of that locus. This distinction is important be-
cause tumor cells frequently duplicate their chro-
mosome complement on a background of LOH
such that one parental allele of a chromosome
is lost, but the other is duplicated. The net ef-
fect is that daughter cells are hemizygous for
a given allele, but retain a normal karyotype
for that particular chromosome. The mechanisms
that cause genomic instability include exposure
to carcinogens, hypoxia, hypomethylation of het-
erochromatic DNA, loss of mitotic checkpoint con-
trols, defective DNA repair, and telomere shortening
[18–20].
Karyotypic studies were the ﬁrst to shed light on
the genetic complexity of cancer pathogenesis, and
one of the ﬁrst observations was that cancer cells of-
ten exhibit signiﬁcant aneuploidy. Solid tumors fre-
quently undergo genome duplication early in their
evolution, and many malignancies exhibit a hy-
potetraploid genotype. Genome duplication occurs
during mitosis, and may involve centrosome am-
pliﬁcation and the formation of multipolar spindles
prior to cytokinesis [21]. Genome duplication prob-
ably occurs in normal cells, but functional mitotic
checkpoints and sentinel DNA damage response
proteins such as p53 and ATM detect aberrant spin-
dle formation and either induce apoptosis or repair
the damage. In preneoplastic cells with mutations
in p53 or other crucial genes, this type of damage
can go undetected.
Karyotypic studies also yielded the ﬁrst informa-
tion about large genetic alterations in lung cancer.
A major step to achieve lung cancer chromosome
analysis occurred with the ability to grow lung
cancer cells in tissue culture, which allowed prepa-
ration of cancer cell metaphases for analysis [22].
Indeed, karyotypic studieswhere the ﬁrst to demon-
strate genetic similarities and differences between
NSCLC and SCLC [23]. Frequent sites of chromoso-
mal losses in SCLC include 3p, 5q, 13q, and 17p.
These occur together with double minutes asso-
ciated with ampliﬁcation of the myelocytomatosis
viral oncogene homolog (MYC), particularly the c-
Myc, family of genes. In NSCLCs, deletions of 3p,
9q, and 17p; +7, i(5)(p10), and i(8)(q10) are com-
mon [23]. Molecular cytogenetic methods includ-
ing array-based comparative genomic hybridization
(CGH), microsatellite marker analysis, and single-
nucleotide polymorphism (SNP) studies have con-
ﬁrmed and extended earlier work. CGH analysis
incorporates whole genome-scale analyses with
relatively high-resolution quantitative information
and revealed gains in 5p, 1q24, and Xq26, and dele-
tions in 22q12.1–13.1, 10q26, and 16p11.2 [23].
Comparative genomic studies led to the ﬁnding
that nearly all SCLCs and many NSCLCs suffer LOHon chromosome 3p, suggesting the presence of one
or more tumor suppressor genes in this chromo-
some region. Although LOH by itself is not sufﬁcient
to indicate the presence of a tumor suppressor lo-
cus, subsequent, high-resolution analyses showed
that in some SCLCs, several genes in the minimally
deleted 3p21.3 and 3p14.2were deleted on both the
maternal and paternal alleles and thus completely
gone from the cancer cell genome, a so-called ho-
mozygous deletion [24]. Homozygous deletions are
rare in cancer cell genomes, and are taken as a
strong indication that a tumor suppressor gene ex-
ists in the affected region. Other homozygous dele-
tions common in lung cancer occur on 9p21 and
17p13. These loci turned out to include the tumor
suppressor genes p16 and p53, respectively. Subse-
quentwork showed the 3p14.2 region to include the
TSG fragile histidine triad, FHIT, while the 3p21.3
region encodes several closely linked TSGs includ-
ing RASSF1A, FUS1, NPRG2, 101F6, SEMA3B, and
SEMA3F [24,25].
Another common type of genomic instability in
lung cancer primarily affects short repetitive se-
quences of DNA, which are called microsatellites.
These microsatellites, while polymorphic can un-
dergo tumor-speciﬁc (compared to normal DNA
from the same patient) alterations in length as a
result of insertion or deletion of the repeating units.
Tumors vary signiﬁcantly in the rate of microsatel-
lite instability (MSI), which may be due to differ-
ences in extant DNA repair pathways as is the case
in colon cancer [17]. MSI can be measured by us-
ing a series of microsatellite markers in polymerase
chain reaction (PCR)-based assays. The overall fre-
quencies of MSI from 13 studies are 35%for SCLCs
and 22% for NSCLCs [26]. However, it remains to
be determined whether MSI is a cause or corollary
of lung tumorigenesis.

Molecular genetics of lung cancer

2009-03-30T15:10:00.001-07:00

Tobacco smoke and lung cancer
It is well known that tobacco smoke is the major
cause of lung cancer. Smokers are 14-fold more
likely to develop lung cancer than nonsmokers
[14]. There are more than 60 carcinogens in to-
bacco smoke, many of which are activated by the
p450 enzymes in the cytosol and then interact co-
valently with DNA, forming DNA adducts [15].
Human cells have evolved specialized mechanisms
that repair different types of DNA adducts, as well
as a speciﬁc DNA polymerase (DNA polymerase)
that can bypass the most common types of DNA
mutations. Benzopyrene and 5-methylchrysene (as
well as other components of tobacco smoke)
form large adducts that cannot be bypassed by
DNA polymerase eta, and need to be removedby nucleotide excision repair (NER). Enzymes in
this DNA repair pathway remove large adducts
by cleaving the DNA helix where adducts have
formed, replacing the affected base, and then ligat
the broken DNA chain. This pathway also repairs
inter- and intra-strand DNA cross-links. Another
important family of tobacco smoke carcinogens, the
N-nitrosamines, frequently induce miscoding mu-
tations. The most common type of miscoding mu-
tation involves alkylation of guanine at the 6O
position, and 6O-methylguanine methyltransferase
repairs this particular change.
Several studies have explored the nature of to-
bacco smoke-induced mutations in lung cancer pa-
tients and have found that themost common type of
mutation is a G-T transversion. When the proﬁle of
tumor-acquired point mutations in the p53 tumor
suppressor gene is compared between smokers and
nonsmokers with lung cancer, there are clear dis-
tinctions in the position and type of mutation that
occur. In smokers, the most frequent type of muta-
tion is G:C>T:A transversion, whereas in lung can-
cer patients with no smoking history, themost com-
mon type of mutation is G:C>A:T transition at CpG
sites (the cytosine in the CpG dinucleotides is partic-
ularly susceptible to spontaneous deamination re-
sulting in a conversion to thymine) [15,16]. Fur-
ther distinctions are apparent when the positions of
G-T transversions are compared between smoking-
related lung cancer and other common types of can-
cer [15].

Overview of lung cancer etiology, incidence, and treatment

2009-03-30T15:09:00.002-07:00

Pathologists have described various different his-
tologies of lung cancer. There are two major sub-
types: small cell lung cancer (SCLC), and nonsmall
cell lung cancer (NSCLC). SCLC accounts for 25%of
lung cancer cases in the United States, and NSCLC
accounts for the remaining 75%. NSCLCs can be
further subdivided into several subtypes: adenocar-
cinoma (Ad), squamous cell carcinoma (SCC), large
cell carcinoma (LCC), bronchioalveolar carcinoma
(BAC), and variousmixed subtypes.While this clas-
siﬁcation system is based on histology, there are sig-
niﬁcant molecular differences between SCLC and
NSCLC. Thus, there is an ongoing effort to describe
these differences in terms of mRNA expression pro-
ﬁles as well as the acquired genetic and epigenetic
changes between the different subtypes. There are
signiﬁcant clinical differences in terms of prognosis
and treatment strategies for the different subtypes.
Therefore, another effort is directed at determin-
ing if speciﬁc molecular abnormalities predict stage
and prognosis as well as the different responses to
chemotherapy and radiation therapy well described
in patients.
We need to understand the molecular differences
between tumors arising in current smokers, for-
mer smokers, and lifetime never smokers. Are there
different acquired molecular abnormalities in lung
cancers arising in women and men or in persons of
different ethnicity or age? Can we use the molec-
ular abnormalities found in lung tissue, sputum,or those shed into the blood as aids for very early
diagnosis or learning who is at the highest risk for
developing cancer? These patients would be can-
didates for extensive screening and early detection
efforts. Could some of the changes be targets for de-
veloping tumor-speciﬁc vaccines or targeting drugs
to speciﬁc molecular abnormalities for therapeutic
purposes? A molecular diagnostic platform was re-
cently approved for use in breast cancer, and similar
designsmust be developed for use in suspected lung
cancer cases. Possible targets for these platforms in-
clude altered gene expression patterns, serum pro-
tein proﬁles, and aberrantly methylated DNA.
Although it seems intuitive that the large mass
of tumor cells that make up the bulk of the tu-
mor should be the target of cancer drugs, recent
evidence suggests that this bulk tumor cell popu-
lation may be less important to tumor progression
than a rare cancer stem cell that can self-renew,
initiate invasion, and propagate metastases. These
cancer stem cells are often less sensitive to cytotoxic
chemotherapy than the bulk primary tumor, and
evade ﬁrst-line therapy as a result. The key to tar-
geting these rare cells is the development of molec-
ularly targeted therapies based on the proﬁle of the
individual tumors.
Individual tumors exhibit signiﬁcant phenotypic
and epigenetic variation, yet they are normally
clonal with respect to crucial genetic alterations.
This means that the evolution of a particular tumor
is driven, at least in part, by the oncogenic changes it
has acquired, and suggests that the continued prop-
agation of the tumor also depends on the activ-
ity of the oncogenes it contains. Bernard Weinstein
likened this effect of oncogene dependence to an
Achilles “heal” for the tumor: he proposed that be-
cause tumors are “addicted” to the presence of a par-
ticular oncogene, they might be uniquely sensitive
to compounds or natural products (antibodies) that
speciﬁcally target the function of the activated onco-
gene [10]. Similarly, a given tumor with a mutation
in a “gatekeeper” tumor suppressor gene whose loss
of function is absolutely required for a particular tu-
mor to develop may be hypersensitive to replacing
the activity of that tumor suppressor gene (TSG).
These concepts are the basis for rational, or molec-
ularly targeted, therapeutic approaches. Severalexamples of this new therapeutic approach are now
available and are having a signiﬁcant impact in the
clinic (Table 4.1).
To fully exploit the potential targets in human
cancer cells for rational drug design, an understand-
ing of the mutational repertoire of human cancer
is necessary. Recently there have been several re-
ports on large-scale sequencing of candidate genes
in cancer cells (such as all tyrosine kinases) that
have identiﬁedmutations in several genes that drug
targets such as PI3 kinases [12]. Following this, the
NIH, in collaborationwith the Broad Institute atMIT
and Johns Hopkins University among others, has
begun to collect data for The Cancer Genome At-
las (TCGA). Over the next decade, this project will
produce a wealth of information that will need to
be analyzed and put into biological context to be
exploited for pharmaceutical development [13].

CHAPTER 4 The Molecular Genetics of Lung Cancer

2009-03-30T15:09:00.001-07:00

A brief history of cancer genetics
That cancer is a genetic disease was ﬁrst understood
near the turn of the last century [1]. After the dis-
covery that DNA was the genetic material, cytoge-
netic studies showed that neoplasms were nearly
always clonal with respect to karyotype and chro-
mosomal pattern. These early genetic studies led to
the concept of the clonal inheritance of somatically
acquired genetic abnormalities in cancer pathogen-
esis [2].
By the late 1960s, there were two competing hy-
potheses both of which derived from the observa-
tion that retroviral-like sequences of DNA and RNA
were frequently found in tumor cells: the idea popu-
larized by Temin involved retrotranscription of viral
genes into host cell DNA (this hypothesis eventu-
ally led to a Nobel Prize for Temin and his postdoc-
toral researcherDavid Baltimore); the other idea de-
veloped by Huebner and Todaro led to the concept
of the oncogene—genes that promote the develop-
ment of cancer [3,4]. The distinction between these
two ideas was the source of the oncogenic element:
in Temin’s view, the carcinogen derived from an in-
fectious agent,whereas for Todaro and Huebner, the
source was an endogenous, vertically transmitted,
retroviral-like gene. Both proposals turned out to
partially correct. By directly testing these compet-
ing hypotheses,Nobel laureates, Varmus and Bishop
were able to show that normal cells contain gene
sequences that are homologous to viral oncogenes;
these sequences are “proto-oncogenes” ready to be
activated during cancer pathogenesis.
Around the same time Alfred Knudson used
statistical inference to devise the complementary
concept of recessive anti-oncogenes. In a classic pa-
per, Knudson postulated that if the overall muta-
tion rate between patients with the inherited form
of retinoblastoma versus the sporadic version were
similar, then the frequent incidence of multifocal
or bilateral retinoblastomas in familial cases must
occur on the background of a germline mutation
in a critical gene [5]. The implication of this study
was that, at least in retinoblastoma, two mutations
were sufﬁcient for the onset of disease: the so-called
“two-hit hypothesis.” Several years later, the gene
that is responsible for familial retinoblastoma was
cloned [6]. In the two decades since retinoblastoma
was ﬁrst cloned and characterized, several hundred
genes—either oncogenes or tumor suppressors or
their accomplices—have been implicated in cancer
pathogenesis [7,8].
The brief overview presented above suggests that
there are at least two distinct genetic components
to cellular transformation: there are large, clonal
chromosome aberrations (aneuploidy) including
translocations, ampliﬁcations, and deletions, and
there are alterations that occur at the level of
the gene, which often include point mutations,
small ampliﬁcations, and deletions. By studying the
genetic lesions that frequently occur in primary
tumor material, cancer geneticists have made sig-
niﬁcant inroads into a general understanding of the
mechanisms of cancer pathogenesis [9]. Modern
molecular biology techniques including cloning, the
polymerase chain reaction, and genome-wide DNA
Lung Cancer, 3rd edition. Edited by Jack A. Roth, James D. Cox,
and Waun Ki Hong. c 2008 Blackwell Publishing,
ISBN: 978-1-4051-5112-2.microarrays have increased the rate with which
new genes are discovered and disease associations
determined. The next step in the biotechnology
revolution will be to translate our growing under-
standing of cancer genetics into rational diagnos-
tic and drug development platforms, and ultimately
into better treatment strategies. This chapter dis-
cusses the genetic basis of lung cancer in light of the
ongoing translational and clinical challenges these
diseases present to physicians, and describes new
approaches to developingmolecularly targeted ther-
apies to treating lung cancer.

References

2009-03-30T15:08:00.001-07:00

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58.

LC risk assessment models

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Statistical models relating multiple risk factors to
cancer risk can identify high-risk subsets of smokers.
There are three criteria to evaluate the performance
of risk assessment models: calibration (reliability),
discrimination, and accuracy [280]. Calibration as-
sesses the ability of a model to predict the num-
ber of endpoint events in subgroups of the popula-
tion and is evaluated by using the goodness-of-ﬁt
statistic. Discrimination is a measure of a model’s
ability to distinguish between those who will and
will not develop disease, and is quantiﬁed by cal-
culating the concordance statistic, or area under a
receiver operating characteristic (ROC) curve. Ac-
curacy including positive and negative predictive
values refers to themodel’s ability to categorize spe-
ciﬁc individuals. The best-known cancer prediction
model is the Gail model for breast cancer [281]. It
has been validated in several populations [282–285]
and appears to give accurate predictions for women
undergoing routine mammographic screening but
probably overestimates the risk for young women
not undergoing routine mammography [286]. The
modest discrimination ability of the Gail model calls
for the incorporation of promising biological fac-
tors [287–290]. Prediction models for other cancers
(melanoma [291,292], colorectal cancer [293], and
LC [7,8]) have also emerged.
The few published LC risk assessment models
mainly focus on smoking behavior and demo-
graphic characteristics. Bach et al. [8] used data col-
lected from CARET, a large, randomized trial of LC
prevention, to derive a LC risk prediction model.
The model used the subject’s age, sex, asbestos ex-
posure history, and smoking history to predict LCrisk andwas derived by use of data fromﬁve CARET
study sites and then validated by assessing the ex-
tent it could predict events in the sixth study site.
The model was then applied to evaluate the risk of
LC among smokers enrolled in a study of LC screen-
ing with computed tomography (CT). The model
identiﬁed smoking variables (duration of smoking,
average number of cigarettes smoked per day, dura-
tion of abstinence), age, asbestos exposure and the
study drug, β-carotene and retinyl palmitate as sig-
niﬁcant predictors of LC. Themodel provided strong
evidence that LC risk varies greatly among smok-
ers and was internally validated and well calibrated
with a cross-validated concordance index of 0.72.
Bach’s model is most applicable to heavy smokers
aged between 50 and 75 years. Recently, Spitz et al.
[294] developed lung cancer risk models for never,
former, and current smokers, respectively. In their
models, factorswith strong etiological roles, e.g., en-
vironmental tobacco smoke, family history of can-
cer, dust exposure, prior respiratory disease, and
smoking history variables were all identiﬁed as sig-
niﬁcant predictors of lung cancer risk. The models
were internally validated with cross-validated con-
cordance statistics for the never, former, and current
smoker models of 0.57, 0.63, and 0.58, respectively.
The computed 1-year absolute risk of lung cancer
for a hypotheticalmale current smokerwith an esti-
mated relative risk close to 9was 8.68%. The ordinal
risk index performed well in that true-positive rates
in the designated high-risk categories were 69%
and 70% for current and former smokers, respec-
tively. When externally validated, this risk assess-
ment procedure could use easily obtained clinical
information to identify individualswhomay beneﬁt
from increased screening surveillance for lung can-
cer. In summary, current LC risk prediction models
have been focused on smoking variables and there
is potential to developmore accuratemodels by col-
lecting more data and incorporating additional risk
factors. Moreover, external validation of existing
models to independent populations is important.Concluding remarks
The results of many reported associations of single
polymorphism analyses are incongruent and couldnot be replicated even with key study parameters
similar to the original ones. Beyond a possible effect
frompopulation heterogeneity, shortcomings in ex-
perimental design and statistical methodology such
as small sample size, lack of control for confound-
ing, selection bias, and multiple comparisons may
account for a large part of these discrepancies. Since
cancer is a multistep and multifactorial disease, the
inﬂuence of individual variants identiﬁed frommost
candidate gene approach studies on overall cancer
riskmight beminimal.Moreover,many cancer risk-
associated genetic variants lack functional valida-
tion. To circumvent these caveats, pathway-based
approaches have been exploited that simultane-
ously analyze the impact of multiple variants in
the same carcinogenesis-related signaling or func-
tion pathway on cancer predisposition. This strategy
might amplify the effect from single variants; how-
ever, the pathway-based approach also depends on
a priori knowledge from basic investigations sug-
gesting the involvement of the pathway in tumori-
genesis. A haplotype-based genome scan approach
has also been proposed to identify causal variants
in the whole-genome scale without any presump-
tion based on prior knowledge, as has been success-
fully applied to isolate causal polymorphisms in a
variety of common human diseases. This approach
mandates stringent study designs, adequate sample
size, and statistical power. In addition, high-power
computational methodologies of data analysis and
error shooting should be developed to probe the vast
amount of interactions amongst genetic and envi-
ronmental factors, and molecular function assays
should be carried out to determine the genotype–
phenotype correlations and validate the biological
signiﬁcance of the identiﬁed high risk alleles.

Growth factor

2009-03-30T15:06:00.000-07:00

IGFs
Both insulin growth factors (IFG1 and IGF2) play
a major role in fostering cell proliferation, survival,
migration and inhibiting apoptosis [264]. The inter-
actions between IGFs and IGFRs are regulated by
IGF binding proteins (IGFBPs) functioning in both
IGF-dependent and IGF-independent manners to
regulate cellular growth [265]. High plasma levels
of IGF1 were associated with increased risk of LC in
a dose-dependent manner [266]. To date, only two
SNPs were reported to predispose to LC. The ho-
mozygous variant at the −202 nucleotide position
of IGFBP3 promoter region was reported to be neg-
atively correlated to LC susceptibility in a Korean
population [267]. This was supported by the ob-
servation that IGFBP3 may have a dual role in the
biosynthesis of IGFs [268], and serum-circulating
IGFBP3 protein might prolong the half-life of IGF
through inﬂuencing its interaction with the mem-
brane receptors [269]. A recent study evaluating
1476 nsSNPs of cancer-related genes identiﬁed a sig-
niﬁcantly altered LC risk associated with Trp138Arg
of IGFBP5 [270]. Using the Pathway Assist software,
11 out of 1476 SNPs exhibiting signiﬁcant LC risk
association were mapped to the GH–IGF axis [270],
indicating the importance of this pathway in LC
development.
EGF
Epidermal growth factor (EGF), a small molecule
ligand that activates receptor tyrosine kinase (RTK),
mediates signal transduction pathways. An A to G
transition in the 5
UTR of EGF gene has been asso-
ciated with reduced LC risk in a Korean population
[271].
VEGF
Vascular endothelial growth factor (VEGF) is a
proangiogenesis protein implicated in carcinogene-
sis and metastasis of many cancers. Three common
polymorphisms in the promoter region (−634G>C,
−1154G>A, and −2578C>A) regulate VEGF pro-
tein level, vascular density, aswell as vascularization
status of tumor tissues from NSCLC patients [272].
However, no study has assessed their implications
in LC risk.Methylation-related genes
Aberrant methylation of pivotal cell growth-related
genesmay lead to carcinogenesis through regulating
their protein expression and common genetic vari-
ants in methylation maintenance genes may also
impact cancer susceptibility.DNMT 3B
DNMT3B is responsible for the generation of ge-
nomicmethylation patterns. To date, three DNMT3B
polymorphisms have been evaluated in LC suscep-
tibility. Wang et al. reported that a C to T single
base substitution in the promoter region was as-
sociated with enhanced promoter activity [273].
Genotypes encompassing the variant allele were as-
sociated with a 1.88-fold excess of LC risk com-
pared to the common homozygotes in Caucasians
[274]. In a Korean population, Lee et al. noted that
the variant alleles of another two promoter poly-
morphisms (−283C>T and −579G>T) were both
associated with reduced risk for LC [275]. The re-
sults of the these studies were in concordance as
both reported that the allele leading to enhanced
DNMT3B expression was associated with increased
cancer risk.
MBD1
MBD1 is a mediator of the DNA methylation-
induced gene silencing. Jang et al. reported that
the wild-type allele of a −634G>A SNP in the pro-
moter region was associated with LC risk with OR
of 3.10 (1.24–7.75) in a Korean population [276].
For another two SNPs (−501delT and Pro401Ala),
the wild-type alleles were correlated with increased
risk of adenocarcinoma but not with other LC
subtypes. Luciferase assays demonstrated that the
haplotype containing the risk-conferring alleles ex-
hibited higher promoter activity, indicating the
presence of a negative correlation between MBD1
expression and LC development.
MTHFR
MTHFR gene encodes an essential enzyme involved
in the production of the S-adenosylmethionine in-
termediate for DNA methylation [277]. Besides a
role in DNA methylation, MTHFR is also important
in maintaining normal cellular folate levels. So far,
two nsSNPs (677C>T and 1298A>C) have been as-
sessed in studies with inconsistent results [278].
SUV39H2
Suppressor of variegation 3–9 homolog 2
(SUV39H2) is a site-speciﬁc histone methyl-
transferase responsible for the methylation oflysine 9 in histone 3. A1624G>C SNP in the 3
UTR region was associated with a 2.63-fold (1.10–
6.29) increased risk of LC in ever smokers when
variant-containing genotypes were compared to
homozygous wild-types [279]. In vitro assays
showed a more than 2-fold higher transcript level
for the variant allele, indicating that this SNP
might be the causal agent functioning through
inﬂuencing protein expression.

Tumor microenvironment

2009-03-30T15:05:00.000-07:00

Microenvironmental factors
Matrix metalloproteins (MMPs) degrade a range of
extracellular matrix and nonmatrix proteins. Since
MMP expression level has been implicated in can-
cer development, several polymorphisms in the pro-
moter regions of MMPs that might affect gene ex-
pression have been evaluated.
MMP1
MMP1 is a highly expressed interstitial collagenase,
which degrades ﬁbrillar collagens. MMP1 is upregu-
lated by tobacco exposure [226] and overexpression
of MMP1 in tumors has been linked to tumor inva-
sion and metastasis [227,228]. A 1G/2G polymor-
phism in the MMP1 promoter was associated with
altered gene expression [229]. Promoters contain-
ing the 2G allele displayed higher transcriptional ac-
tivity than those with the 1G allele. An increased LC
risk was identiﬁed with the 2G/2G genotype by Zhu
et al. [230]. Two other studies, Su et al. [231] and
Fang et al. [232] both noted an increase in LC risk
with the 2G allele, but this did not reach statistical
signiﬁcance.
MMP2
MMP2 is a gelatinase whose substrates include
gelatins, collagens, and ﬁbronectin. The expression
levels of MMP2 have been commonly used to pre-
dict cancer prognosis. A promoter SNP (−1306C>T)
has been associated with reduced activity due to
a possible interference with the SP1-binding site
[233]. Interestingly, compared to the variant allele
associated with lower gene expression, the wild-
type allele exhibited an association with LC risk
with an OR of 2.18 (1.70–2.79) in a Chinese pop-
ulation [234]. Another promoter SNP (−735C>T),
which was linked with −1306C>T, was also associ-
ated with risk for the wild-type allele with an OR of
1.57 (1.27–1.95) [235] which retains the SP1 bind-
ing site as well as a higher transcriptional activa-
tion efﬁciency [236].Moreover, itwas noted that an
even higher risk was associated with the haplotype
containing the wild-type alleles at both loci and this
risk showed a multiplicative interaction effect with
smoking [235].
MMP3
MMP3 is a stromelysin whose substrates include
collagens, gelatin, aggrecan, ﬁbronectin, laminin,
and casein. The most commonly studied MMP3
polymorphism is a promoter variant located at
−1171 nucleotide, containing either ﬁve or six
adenosines that may affect promoter transcription
activity [237]. In a Caucasian population, a hap-
lotype containing the 6A allele exhibited a higher
LC risk in never smokers [238]. However, in a Chi-
nese study, Fang et al. reported that smokers with
the MMP3 5A allele had a 1.68-fold (1.04–2.70) in-
creased risk to develop NSCLC [232].
MMP7
MMP7 is a matrilysin whose substrates include col-
lagens, aggrecan, decorin, ﬁbronectin, elastin, and
casein.MMP7 is highly expressed in lungs of patients
with pulmonary ﬁbrosis and other conditions asso-
ciated with airway and alveolar injury. The variant
allele of a promoter SNP, –181A>G, might lead to
higher promoter activity and increased mRNA lev-
els [239]. Consistently, the variant-harboring geno-
types, when compared to the common homozy-
gotes, have been proven to predispose to risk of
NSCLC [240].
MMP9
MMP9 is a gelatinase and the major structural com-
ponent of the basement membrane. Hu et al. re-
ported that two common nsSNPs, Arg279Gln and
Pro574Arg,might confer LC susceptibility in a dose-
dependent fashion [241].
MMP12
MMP12 is a metalloelastase required for
macrophage-mediated extracellular matrix pro-
teolysis and tissue invasion. A promoter SNP
(−82A>G) might regulate MMP12 expression
through modulating the binding afﬁnity of tran-
scription activation protein 1 [242]. Another
nsSNP (1082A>G) leads to the substitution of
serine for asparagine. Although no signiﬁcant LC
risk associations were identiﬁed for either SNP, a
haplotype containing the −82A and 1082G alleles
was associated with higher LC risk among never
smokers in comparison to haplotypes containing
−82G and 1082A [238].
Inﬂammation
Airway inﬂammation may promote tumorigenesis
through multiple mechanisms such as inducing ox-
idative stress and lipid peroxidation [243]. To date,
only a few polymorphisms in inﬂammation genes
have been evaluated for their roles in lung tumori-
genesis and the results have beenmostly discrepant.
Anti-inﬂammatory genes
The major functions of anti-inﬂammation genes
such as IL4, IL10, IL13, and PPARs are to resolve the
acute inﬂammatory reactions. Among these, IL10 is
produced by monocytes and lymphocytes and ex-
hibits multiple functions in the regulation of cell-
mediated immunity, inﬂammation, and angiogene-
sis [244]. Three SNPs in the promoter region of IL10
have been identiﬁed (−1082A>G, −819C>T, and
−592C>A). In a Chinese study, the variant allele
of the −1082A>G SNP was associated with a 5.26-
fold (2.65–10.4) increased LC risk [245], which was
in agreement with another study in small cell LC
[246]. The variant allele has been shown to affect
IL10 protein level through regulating gene tran-
scription [247–249].
Proinﬂammatory genes
Engels et al. [250] systematically evaluated a panel
of 59 single nucleotide polymorphisms (SNP) in 37
inﬂammation-related genes among non-Hispanic
Caucasian lung cancer cases (N = 1,553) and con-
trols (N = 1,730). They found that Interleukin 1
beta (IL1B) C3954T was associated with increased
risk of lung cancer and that one IL1A-IL1B hap-
lotype, containing only the IL1B 3954T allele, was
associated with elevated lung cancer risk. These as-
sociations were stronger in heavy smokers, partic-
ularly for IL 1B C3954T. IL1B activates a mixture
of inﬂammatory signaling mediators including NF
Kappa B, leading to an ampliﬁed proinﬂammatory
effect. A variable number of tandemrepeats (VNTR)
polymorphism in intron 2 of the IL1RN gene [251]
inﬂuences the expression of both IL1B and IL1RN
[252,253]. Lind et al. [251] observed an increased LC
risk in individuals with both the IL1RN∗
1 and the

Apoptosis

2009-03-30T15:04:00.000-07:00

Genetic polymorphisms in
apoptotic pathways
Apoptosis (programmed cell death) is an essential
cellular defense mechanism. Two principal signal-
ing pathways, the intrinsic pathway and the ex-
trinsic pathway, are implicated in the coordination
of the apoptotic process. In the extrinsic apopto-
sis pathway, polymorphisms inﬂuencing the FASL–
FAS interaction might affect LC predisposition. In
a Chinese case–control study, two promoter SNPs
of FAS (−1377G >A) and FASL (−844T >C) [219]
were associated with increased LC risks with ORs
of 1.59 (1.21–2.10) and 1.79 (1.26–2.52), respec-
tively, when the rare homozygotes were compared
to the common homozygotes. A multiplicative in-
teractive effect was noted. In the intrinsic apop-
tosis pathway, CASP9 is the only gene that has
been assessed for a role in LC development. In a
Korean study, Park et al. found that two CASP9
promoter SNPs (−1263A >G and −712C >T) were
associated with signiﬁcantly altered LC risk with
OR’s of 0.64 (0.42–0.98) and 2.32 (1.09–4.94),
respectively, when their homozygous variant geno-
types were compared to the homozygous wild-type
reference group [220]. Furthermore, the haplo-
type composed of the G allele of −1263A >G and
the C allele of −712C>T was associated with a
signiﬁcantly decreased risk. This was consistent
with the results from single SNP analysis and
was functionally validated by a promoter-luciferase
assay.
Phenotypic assays in apoptotic pathways
Two groups have reported that impaired mutagen-
induced apoptotic capacity was associated with in-
creased risk of LC [217] using the TUNEL (Terminal
transferase dUTP nick end labeling) method [221].
Biros et al. [192] reported that in LC patients, indi-
viduals with the variant allele of p53 Pro72Arg poly-
morphismexhibited a lower level of apoptoticwhite
blood cells. This ﬁnding was consistent with the re-
sults fromWu et al. [187] who showed that the hap-
lotype containing the wild-type alleles of the three
p53 polymorphisms (intron 3, exon 4, and intron
6) was associated with higher apoptotic index than
those with at least one variant allele.
Telomere and telomerase
Telomeres are TTAGGG repeat complexes bound by
specialized nucleoproteins at the ends of chromo-
somes in eukaryotic cells. By capping the ends of
chromosomes, telomeres prevent nucleolytic degra-
dation, end-to-end fusion, irregular recombination
and other events lethal to cells [222].Wu et al. [223]
measured telomere length in the peripheral blood
lymphocytes of LC patients and age-matched con-
trols and found signiﬁcantly shorter telomere length
in LC cases.
To date, only two studies have indicated that com-
mon sequence variants in the TERT genomic region
might predispose to LC. TERT is the protein moi-
ety of telomerase, the key enzyme in the mainte-
nance of telomere length by synthesizing TTAGGG
nucleotide repeats. In the majority of human can-
cers, telomerase is activated and cells overcome
senescence and become immortalized. Wang et al.
[224] showed that a polymorphic tandem repeat
minisatellite (MNS16A) in the promoter region of
an antisense transcript of the TERT gene regulates
the antisense transcript expression. Cells with short
tandem repeats displayed higher promoter activity
compared to those with longer tandem repeats. A
subsequent case–control study showed that the long
tandem repeat variant was associated with a more
than 2-fold increase in LC risk in a recessive pat-
tern, supporting the conjecture that the antisense
transcript might serve as a tumor suppressor gene
inhibiting the expression of TERT [224]. Another
polymorphism was recently identiﬁed in the Ets2
binding site of the TERT promoter region [225].
Compared to the common homozygotes, the rare
homozygotes exhibited reduced telomerase activity.

Genetic variations in LC risk assessment 4

2009-03-30T15:03:00.000-07:00

ATM
In human cells, ATM is required for the early re-
sponse to ionizing radiation. ATM senses genomic
damage and initiates DNA repair through interact-
ing with the MRN (MRE11, RAD50, and NBS1)
complex and subsequently activating a series of
downstream signaling mediators [153]. In a Korean
study, an SNP in intron 62 (IVS62 +50G>A) ex-
hibited a signiﬁcantly increased LC risk with an OR
of 1.6 (1.1–2.1) [154], and higher risks for haplo-
types and diplotypes containing the variant allele
with ORs of 7.6 (1.7–33.5) and 13.2 (3.1–56.1), re-
spectively. The close proximity of this SNP to ATM
PI3K and FAT domains suggests a potential func-
tional impact on ATM kinase activity.
NBS1
In the HR pathway, the ﬁrst event is the resec-
tion of the DNA to yield single-strand overhangs
[118]. NBS1 is part of an exonuclease complex that
takes part in this step. Zienolddiny et al. and Mat-
ullo et al. showed no association between the NBS1
Glu185Gln polymorphism and LC risk [125,155],
but Lan et al. reported that homozygotes for this al-
lele had an increased risk of LC with an OR of 2.53
(1.05–6.08) [156].
XRCC3
XRCC3 is an RAD51-related protein involved in cat-
alyzing the DNA strand exchange reaction during
HR [118]. Five epidemiological studies found no
association between the thr241Met polymorphism
with LC risk [125,143,148,155,157].
LIG4
In the NHEJ pathway, LIG4 has an important role in
linking the ends of a double-strand break together
[118]. Matullo et al. found no association between
two LIG4 polymorphisms (Ala3Val and Thr9Ile) and
LC risk [155]. Sakiyama et al. reported that the vari-
ant allele of the Ile658Val polymorphism of LIG4
was associated with a reduced risk of squamous cell
carcinoma with an OR of 0.4 (0.1–0.8) [158].
MMR
The MMR system maintains the stability of the
genome during repeated duplication, by repairing
base–base mismatches, caused not only by errors
of DNA polymerases that escape their proofreading
function, but also by insertion/deletion loops that
result from slippage during replication of repetitive
sequences or during recombination [118]. So far,
only a few studies have investigated the connection
between MMR and LC.
MLH1 –93G>A polymorphism was studied for its
association with risk of LC and no overall associa-
tionwas identiﬁed [159]; Jung et al. investigated the
association of MSH2 –118T>C, IVS1 +9G>C, IVS10
+12A>G, and IVS12 –6T>C genotypes with LC risk
[160] and found that the presence of at least one
IVS10 +12G allele was associated with a decreased
risk of adenocarcinoma as compared with the IVS10
+12AA genotype with OR of 0.59 (0.40–0.88), and
the presence of at least one IVS12 –6C allele was as-
sociated with an increased risk of adenocarcinoma
as compared with the IVS12 –6TT genotype with an
OR of 1.52 (1.02–2.27) [160].
DNA damage and repair phenotypic assays
The phenotypic assays for DNA damage and repair
include measuring: (a) DNA damage/repair after a
chemical or physicalmutagen challenge (such as the
mutagen sensitivity, comet, and induced adduct as-
says); (b) unscheduled DNA synthesis; (c) cellular
ability to remove DNA lesions from plasmid trans-
fected into lymphocyte cultures in vitro by expres-
sion of damaged reporter genes (the host–cell reac-
tivation assay); (d) activity of DNA repair enzyme
(repair activity assay for 8-OH-Guanine) [161,162].
Mutagen sensitivity
The mutagen sensitivity assay quantiﬁes chromatid
breaks induced by mutagens in cultured lympho-
cytes in vitro as an indirect measure of DRC
[163,164]. Bleomycin is a clastogenic agent that
mimics the effects of radiation by generating free
oxygen radicals capable of producing DNA single-
and double-strand breaks that initiate BER and DSB
repair [165].Wu et al. showed that higher BPDE and
bleomycin sensitivities were independently signiﬁ-
cantly associated with increased risks of LC, a ﬁnd-
ing that has been conﬁrmed by other studies [3–
5,166,167].
Comet assay
The comet assay is a single-cell gel electrophoresis
method used to measure DNA damage in individ-
ual cells. It is a sensitive and versatile method with
high throughout potential [168,169]. The alkaline
version (pH > 13) of the comet assay can detect
DNA damage such as single-strand breaks, double-
strand breaks, and alkaline labile sites [170]. Com-
mon mutagens used in this assay include BPDE,
bleomycin, and γ-radiation. Wu et al. found that
higher γ-radiation- and BPDE-induced olive tail
moments, one of the parameters for measuring
DNA damage, were signiﬁcantly associated with
2.32- and 4.49-fold risks of LC, respectively [171].
Rajaee-Behbahani et al. reported lower repair rate of
bleomycin-induced DNA damage using the alkaline
comet assay in LC patients compared with controls
[172].
DNA adducts
Using 32P postlabeling techniques, two studies by
the same group indicated a signiﬁcant association
between the level of in vitro BPDE-induced DNA
adducts and risk for LC [173,174], suggesting sub-
optimal ability to remove the BPDE-DNA adduct re-
sulted in increased susceptibility to tobacco carcino-
gen exposure [174].
Host cell reactivation assay
The host cell reactivation assaymeasures globalNER
as a biomarker for LC susceptibility [175–177], by
quantifying the activity of a reporter gene (CAT
or LUC gene) in undamaged lymphocytes trans-
fectedwith BPDE-treated plasmids. Because a single
unrepaired BPDE-induced DNA adduct can block
reporter gene transcription [178], the measured
reporter gene activity reﬂects the ability of the trans-
fected cells to remove the adducts fromthe plasmid.
Reduced capacity to repair adducts is observed in
cases compared to controls and is associated with
an increased risk of LC with evidence of a signiﬁ-
cant dose–response association between decreased
DRC and risk of LC [175–177].
8-OGG assay
The enzyme 8-oxoguanine DNA N-glycosylase is
encoded by the OGG1 gene and initiates the BER
pathway. The OGG activity assay monitors the abil-
ity ofOGG to remove an 8-oxoguanine residue from
a radiolabeled synthetic DNA oligonucleotide, gen-
erating two DNA products that can be distinguished
on the basis of size [179]. Paz-Elizur et al. showed
that OGG activity was signiﬁcantly lower in periph-
eral blood mononuclear cells from LC patients than
in those fromcontrols. Individuals in the lowest ter-
tile of OGG activity exhibited an increased risk of
NSCLC compared with those in the highest tertile
(OR=4.8; 95%CI, 1.5–15.9) [179].Gackowski et al.
also reported that the repair activity of OGG was
signiﬁcantly higher in blood leukocytes of healthy
volunteers than in LC patients [180].
Cell cycle control
The intricate cell cycle regulatory network is essen-
tial for cells to undergo replication, division, prolif-
eration, and differentiation. Anomalies of cell cycle
regulation genes are frequently observed in a vari-
ety of human malignancies including LC, and are
considered to be one of the most critical early-stage
events in carcinogenesis [181–184].
Genetic polymorphisms in cell
cycle-related genes
p53
p53 is the most important tumor suppressor gene of
the genome defense system regulating pivotal cel-
lular activities such as DNA damage response, DNA
repair, cell cycle control, and apoptosis. Three poly-
morphisms of the p53 gene have been commonly
studied in cancer susceptibility.Weston et al. ﬁrst re-
ported the association between the Arg72Pro nsSNP
in exon 4 and increased LC risk [185], which was
conﬁrmed by a number of subsequent studies in
various populations [186–191]. Functional assays
corroborated this ﬁnding by demonstrating the as-
sociation between the variant allele and increased
p53 mutations in tumor tissues, as well as a reduced
rate of apoptosis in white blood cells of LC patients
[189,192,193]. Wu et al. reported an association
with the variant genotype of both the intron 3 16-bp
deletion/insertion and the intron 6 polymorphisms
[187]. Analyses of haplotypes reconstructed using
these three polymorphisms demonstrated an in-
creased LC risk for the variant-harboring haplotypes
compared to the haplotype with wild-type alleles at
all three loci. This result was supported by func-
tional studies showing that the variant-harboring
haplotypes exhibited a reduced apoptotic index and
reduced DNA repair capacity [187]. Moreover, the
association with the intron 3 polymorphism was
conﬁrmed in a recent large-scale European study,
which reported a 2.98-fold [194] increased LC risk
for the homozygous variant genotype. Although
most studies suggest a positive association between
these p53 polymorphisms and LC risk, disagree-
ments exist including a recent meta-analysis of
13 LC studies showing no LC risk association for
any of these polymorphisms [195].
p73
p73 may activate p53 down-stream transcriptional
effectors such as p21 to control cell cycle progression
and apoptosis [196]. A dinucleotide polymorphism
in the 5
UTR of p73 is associated with an increased
risk of LC in a Caucasian population but a protec-
tive effect in a Chinese population [197,198], sug-
gesting the possible existence of ethnic-speciﬁc risk
differentiation. Furthermore, a gene–dosage effect
by combining both p53 and p73 variant alleles to-
gether was demonstrated [199].
MDM2
MDM2, a ubiquitin ligase, negatively regulates p53
activity either by binding to the transactivation do-
main of p53 protein and inhibiting its transcrip-
tional activation of p21, or by targeting p53 pro-
tein to ubiquitin-mediated proteasome degradation
[200].AT toGtransversion in the intronic promoter
region of MDM2 was associated with increased LC
risk in Chinese [190], Koreans [201], and Euro-
peans [202]. Other studies exhibited similarly el-
evated risk, although not reaching statistical sig-
niﬁcance [203,204]. The agreement amongst these
studies recapitulates the in vivo observation that
the variant allele upregulates MDM2 expression and
thus reduces p53 protein level [205].
HRAD9
HRAD9 is a phosphorylation target of ATM kinase
that plays a crucial role in DNA repair and cell cycle
arrest in response to DNA damage [206]. A nsSNP

Genetic variations in LC risk assessment 3

2009-03-30T15:02:00.000-07:00

ERCC2/XPD
Two SNPs in the XPD gene have been partic-
ularly well studied: Asp312Asn and Lys751Gln.
Manuguerra et al., in a meta-analysis, showed that
the two homozygote variant genotypes were as-
sociated with increased risk of LC with ORs of
1.25 (1.04–1.51) and 1.24 (1.05–1.47), respectively
[120]. Spitz et al. reported that both polymorphisms
might modulate NER capacity in LC patients [121];
both cases and controlswith thewild-type genotype
exhibited the most proﬁcient DNA repair capacity
(DRC) as measured by the host cell reactivation as-
say. A statistically signiﬁcant difference in allele fre-
quency between different ethnic groups has been
observed for these two SNPs [120].
XPA
A polymorphic site (–4A>G) in the 5
UTR region of
the XPA gene has been evaluated in several studies,
withmost identifying theGallele as being associated
with reduced LC risk [122–124]. One study, how-
ever, suggested that the A allele had a protective role
in LC [125]. Wu et al. reported that control subjects
with one or two copies of the G allele demonstrated
more efﬁcientDRC than did thosewith the homozy-
gous A allele as measured by the host cell reactiva-
tion assay [122]. This ﬁnding is consistent with the
putative association reported with reduced LC risk
in Caucasians.
XPC
XPC binds to HR23B, and the XPC-HR23B com-
plex functions as an early DNA damage detector in
NER [118,119]. Lee et al. studied the association of
seven XPC polymorphisms (–449G>C, –371G>A, –
27G>C, Ala499Val, PAT −/+, IVS11 –5C>A, and
Lys939Gln)with LC risk in a Korean population and
found that only the –27C allele was associated with
a signiﬁcantly increased risk for LC with an OR of
1.97 (1.22–3.17) [126]. The PAT−/+ is a biallelic
poly (AT) insertion/deletion polymorphism in in-
tron 9. PAT+ homozygotes exhibited signiﬁcantly
lower DRC than the wild-type homozygotes [127].
PAT+/+ subjects were at signiﬁcantly increased risk
for LCwithOR of 1.60 (1.01–2.55) in a Spanish pop-
ulation [128]. Hu et al. showed that the minor Val
allele of Ala499Val was associated with increased
risk of LC in a Chinese population [129].
ERCC1
ERCC1, a highly conserved enzyme, is required for
the incision step of NER [118,119]. Two common
polymorphisms, C8092A and Asn118Asn, are as-
sociated with altered ERCC1 mRNA stability and
mRNA levels [130,131]. Zhou et al. reported no
overall association between these two SNPs and LC
risk [132]; however, Zienolddiny et al. showed that
the rare homozygous genotype of Asn118Asn poly-
morphism was associated with a signiﬁcantly in-
creased risk of nonsmall cell LC (NSCLC) (OR =
3.11; 95% CI, 1.82–5.30) [125].
XPG
XPG functions as a structure-speciﬁc endonucle-
ase that cleaves the damaged DNA strand in
NER [118,119]. Two studies investigating the
His1104Asp polymorphismcame to the similar con-
clusion that the rare homozygote genotype had a
protective role in LC [133,134].
BER
In parallel with NER proteins which primarily op-
erate on bulky lesions, the BER proteins mainly
repair damaged DNA bases arising from endoge-
nous oxidative processes and hydrolytic decay of
DNA. OGG1, APE1, and XRCC1 are three key play-
ers in BER. OGG1 is a base-speciﬁc glycosylase that
initiates repair by releasing the modiﬁed base, 8-
oxoguanine, and creating abasic sites. APE1 is an
endonuclease that incises theDNAstrand at the aba-
sic site. XRCC1 functions as a scaffold protein in BER
by bringing DNA polymerase and ligase together at
the site of repair [118]. Genetic polymorphisms in
these three genes have also been implicated in LC
risk.
OGG1
A high incidence of spontaneous lung adenoma
and carcinoma was found in OGG1-knockout mice,
which suggested that OGG1 acts as a suppressor of
LC [135]. Four studies concluded that the homozy-
gous variant genotype of Ser326Cys was associated
with signiﬁcantly increased risk of LC [125,136–
138], while one reported a borderline signiﬁcance
[139]. A meta-analysis also found increased risk
among subjects carrying the homozygous variant
genotype (OR = 1.24; 95% CI, 1.01–1.53) [140].
These ﬁndings are consistent with experimental
evidence that this isoform exhibits lower enzyme
activity [141].
APE1
Four case–control studies investigated the associa-
tion between Asp148Glu SNP and LC risk all report-
ing no signiﬁcant association [125,142–144]. Sub-
jects who had the rare homozygote of Ile64Val had
reduced risk of NSCLC with an OR of 0.10 (0.01–
0.81). No signiﬁcant association was observed for
the Gln51His polymorphism [125].
XRCC1
There are three extensively studied SNPs of
XRCC1, Arg194Trp, Arg280His, and Arg399Gln. The
Arg194Trp variant has been shown to be associated
with BPDE sensitivity in vitro [145]. Most studies
have reported a trend for reduced risk for the Trp al-
lele in LC [125,139,146,147], as did a meta-analysis
of tobacco-related cancerswith anOR of 0.86 (0.77–
0.95), based on 4895 cases and 5977 controls from
16 studies [140]. The results for the Arg280His poly-
morphism were contradictory [139,143,146]. Most
studies showed no signiﬁcant association between
Arg399Gln and LC risk [125,139,142–144,146,148–
151] and this ﬁnding has been conﬁrmed by a
meta-analysis with 6120 LC cases and 6895 controls
[152].
DSB
DSB is considered the most detrimental to cellu-
lar DNA damage as both DNA strands are affected.
DSBs arise froma number ofmechanisms, including
ionizing radiation, X-ray, certain chemotherapeutic
agents and replication errors [118]. Homologous re-
combination (HR) and nonhomologous end-joining
(NHEJ) are two complementary pathways in DSBR.

Genetic variations in LC risk assessment 2

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CYP2C9
CYP2C9 is involved in the activation of PAH
and heterocyclic aromatic amines. The CYP2C9∗
2
(Arg144Cys) is themost intensively studied SNP, but
results have been inconsistent due to small study
sizes [77–79].
CYP2D6
CYP2D6 is responsible for the metabolism of NNK.
In a meta-analysis of 13 studies, no association was
noted with the variant genotype [80]. This was con-
ﬁrmed by studies in different ethnic groups [81,82].
CYP2E1
CYP2E1 metabolizes a wide variety of carcinogens
including NNK. There is an Rsa I polymorphism in
the 5
ﬂanking region which has been shown to af-
fect gene transcription [83], and individuals in var-
ious populations with the variant allele exhibited a
signiﬁcant reduction in LC risk [84–88]. Phenotypic
assays suggest that the variant allele is associated
with impaired host capacity to process chlorzoxa-
zone to its active metabolites [89]. However, a few
studies reported null results for this polymorphism
and LC risk [79]. Similar conﬂicting results were
also reported for the Dra I polymorphism located in
intron 6 [83,90–92].
MPO
Myeloperoxidase (MPO) transforms a broad range
of tobacco smoking-derived procarcinogens such as
B[a]P. A promoter SNP (–463G>A) has been asso-
ciated with reduced gene expression and enzymatic
activity, as well as reduced level of tobacco-derived
DNA adducts [93,94]. Ameta-analysis of 2686 cases
and 3325 controls [95] reported a nonsigniﬁcantly
reduced LC risk associated with the variant allele
with an OR of 0.86 (0.67–1.1). This ﬁnding is con-
sistent with another study [96].
GST family
GST is a family of soluble detoxiﬁcation enzymes
that mainly catalyzes the conjugation of GSH with
intermediate cytotoxic compounds metabolized by
phase I enzymes. There are four classes of GST
enzymes, namely, GST alpha (GSTA), GST mu
(GSTM), GST pi (GSTP), and GST theta (GSTT).
Common variants in the GST family have been ex-
tensively investigated but the results have been in-
consistent. A recent meta-analysis compiling 119
previous studies with 19,729 cases and 25,921 con-
trols reported a 1.18-fold increased LC risk (1.14–
1.23) associated with the null genotype [97]. How-
ever, when the analysis was restricted to ﬁve large
studies (3436 cases and 3897 controls) with >500
cases in each to avoid potential publication bias,
only a slight increase in LC risk was identiﬁed with
an OR of 1.04 (0.95–1.14). For the whole-gene
deletion polymorphism of GSTT1, a meta-analysis
with 9632 cases and 12,322 controls from 44 previ-
ous studies reported a 1.09-fold increase in LC risk
(1.02–1.16) [97]; however, restricting the analysis
to four large studies with >500 cases eliminated this
effect with an OR of 0.99 (0.86–1.11). Publication
bias and study heterogeneity were not detected in
this GSTT1 meta-analysis, suggesting limited poten-
tial of this polymorphism alone as a risk factor for
LC. Two SNPs (Ile105Val and Ala114Val) have been
commonly studied for the GSTP1 gene. A reduction
in GSTP1 enzyme activity has been reported to be
associated with the variant allele of both SNPs [98].
However, ameta-analysis with 6221 cases and 7602
controls from 25 previous studies, and with 1251
cases and 1295 controls from4 studies did not reveal
any signiﬁcant association with LC risk for either
variant [97]. GSTM3 has a 3bp-deletion polymor-
phism in intron 6, which has been investigated in
different populations but again with mixed results.
A meta-analysis of ﬁve studies with 1238 cases and
1179 controls did not note any signiﬁcant associa-
tion with LC risk with an OR of 1.05 (0.89–1.23)
[97].
NAT family
Human N-acetyltransferases (NATs) are generally
classiﬁed into two categories, slow and fast acetyla-
tors, depending on the effect on protein enzymatic
activity by the polymorphisms. So far, the studies of
LC risk with NAT polymorphisms have yielded in-
consistent results. For NAT1, signiﬁcant gene–dose
effects were observed between slow acetylators and
increased risk of LC in one study [99]. Conversely,
a German study [100] demonstrated that the NAT1
fast acetylators had an increased risk of lung
adenocarcinoma, but not squamous cell carcinoma,
which was consistent with the observation that fast
NAT1 acetylators exhibited a higher level of chro-
mosomal aberrations as well as increased LC risk
in smokers [101]. Similarly, discordant conclusions
have been noted for NAT2 polymorphisms with LC
risk.Arecentmeta-analysis of 16 studieswith a total
of 3865 cases and 6077 controls failed to detect any
statistically signiﬁcant difference in NAT2 acetylator
status and LC risk [102].
UGT family
UDP-glucuronosyltransferase (UGT) superfamily
catalyzes the conjugation of a glucuronic acid moi-
ety to the nucleophilic substrate resulting from
the phase I bioactivation. A small Japanese study
showed that the UGT1A7∗
3 polymorphism was as-
sociated with a 4.02-fold (1.57–10.30) increased LC
risk compared with the wild-type UGT1A7∗
1 allele
[103].
SULT family
The sulfotransferase (SULT) family catalyzes the
sulfonation of a wide spectrum of exogenous and
endogenous compounds. SULT1A1 G638A is the
most studied SNP and the variant allele has been
associated with reduced sulfotransferase activity
[104]. Compared with the wild-type, the variant-
containing genotypes exhibited a signiﬁcantly in-
creased LC risk with an OR of 1.85 (1.44–2.37) in a
Chinese population [105], which was in line with a
Caucasian study showing a 1.41-fold increased LC
risk (1.04–1.91) [106].
NQO1
NAD(P)H:quinono oxidoreductase 1 (NQO1) is a
cytosolic protein that metabolizes quinoid com-
pounds and derivatives. In vitro studies demon-
strated a signiﬁcantly reduced enzyme activity asso-
ciated with the variant allele of a Pro187Ser SNP in
a dose-dependent fashion [107,108]. Nonetheless,
a meta-analysis of 19 studies with a total of 6980
cases and 8080 controls reported no signiﬁcant LC
risk association [109].
EPHX1
There are two commonly studied SNPs (Try113His
and His139Arg) in the microsomal epoxide hydro-
lase 1 (EPHX1) gene that encodes a phase II pro-
tein involved in the hydrolysis of reactive aliphatic
and arene epoxides derived from PAH and aromatic
amines [110]. Mixed results were derived from a
number of small case–control studies evaluating
their associations with LC risk. A pooled analysis of
986 cases and 1633 controls from eight studies re-
ported a decreased LC risk associated with the vari-
ant allele of Try113His polymorphism with OR of
0.70 (0.51–0.96) [111],whichwas further validated
by an Austrian study [112]. In both studies, the rare
allele of His139Argwas associatedwith an increased
LC risk that did not reach statistical signiﬁcance.
GPX1
Glutathione peroxidase I (GPX1) is a selenium-
dependent phase II antioxidant enzymewhich plays
a crucial role in detoxifying organic peroxides and
hydrogen peroxides [113]. A nsSNP (Pro198Leu)
has been widely assessed for an association with LC
risk, but the results appear to be conﬂicting in dif-
ferent populations [114–116].
DNA damage and repair
The capacity to repair DNA damage from endoge-
nous and exogenous sources is crucial to maintain-
ing genomic integrity. Nucleotide-excision repair
(NER), base-excision repair (BER), double-strand-
break repair (DSBR), and mismatch repair (MMR)
are four key DNA repair pathways that operate on
different types of DNA damage. Common genetic
variants in genes involved in these repair path-
ways have been reported in many LC susceptibility
studies [117].
Genetic polymorphisms in DNA repair genes
NER
NER is capable of removing a wide class of helix-
distorting lesions that interfere with base pair-
ing and generally disrupt transcription and normal
replication resulting from tobacco smoking such as
benzo[a]pyrene diol-epoxide (BPDE) [118]. NER is
themost versatile DNA repair pathway and includes
∼30 proteins involved in DNA damage recognition,

Genetic variations in LC risk assessment

2009-03-30T15:01:00.001-07:00

Individual susceptibility could be modulated by ge-
netic variants in genes involved in many cellu-
lar processes such as carcinogen metabolism, DNA
repair, cell cycle checkpoint control, apoptosis,
telomere integrity and microenvironment control.
Numerous molecular epidemiological studies have
been conducted to evaluate the associations of com-
mon sequence variants of these genes with LC risk
but the results are conﬂicting for most polymor-
phisms. The following section will focus on some
consistent results as well as those contradictory re-
sults that merit further investigation.
Carcinogen metabolism
Tobacco carcinogens aremetabolized by phase I and
phase II xenobiotic enzymes. Phase I enzymes (the
cytochrome P450 (CYP) family members) are in-
volved in the activation of carcinogens to formelec-
trophilic metabolites that are further processed by
phase II detoxiﬁcation enzymes through the conju-
gation of hydrophobic or electrophilic compounds.
Several groups of enzymes are involved in these
steps.
CYP1A1
CYP1A1 is the most extensively studied phase I car-
cinogen metabolizing enzyme involved in bioacti-
vation of a wide spectrum of carcinogens including
benzo[a]pyrene (B[a]P), one of the most abundant
polycyclic aromatic hydrocarbon (PAH) carcinogens
derived from tobacco-smoking. Two CYP1A1 sin-
gle nucleotide polymorphisms (SNPs) have been as-
sessed in LC association studies. In a meta-analysis
of 22 studies with 1441 Caucasian cases and 1779
controls, when compared to the common homozy-
gotes, the rare homozygote variant T3801C geno-
type carriers had a 2.28-fold increased LC risk (0.98–
5.28) [62]. In a pooled analysis of 11 studies, Le
Marchand et al. noted a dose–response effect of
increasing risk of LC with increasing number of the
variant allele of the CYP1A1 Ile462Val SNP [63].
CYP1B1
CYP1B1 is an extrahepatic xenobiotic enzyme ex-
pressed in the human lung, and is inducible upon
exposure to tobacco smoking and B[a]P [64]. A
nonsynonymous SNP (nsSNP) results in the substi-
tution of leucine by valine in exon 3. Though no sig-
niﬁcant main effects of this polymorphism and LC
risk have been found, subgroup associations have
been reported [65,66].
CYP2A6
CYP2A6 transforms nicotine into 3-hydroxycoti-
nine, the primary urinary nicotine metabo-
lite in tobacco smokers. It is also involved in
the metabolism of nitrosamine 4-(methylnitros-
amino)-1-(3-pyridyl)-1-butanone (NNK), N
-nitro-
sodimethylamine (NNN), and N-nitrosodiethyla-
mine (NDEA), major nitrosamine carcinogens
resulting from tobacco combustion. Considerable
interindividual genetic variation exists in the
CYP2A6 gene, many variants of which have been
associated with altered carcinogen metabolizing ca-
pacity. Particularly interesting, those homozygous
for CYP2A6∗
4C, a whole-gene deletion polymor-
phism[67], exhibited lower LC risk only in smokers
[68–70], supporting the notion that subjects with
reduced activity of phase Imetabolismenzymesmay
have lower cancer risk. Itwas also suggested that in-
dividuals with the variant allele and hence reduced
CYP2A6 activity are less likely to become addicted
to nicotine, making it is easier to quit smoking and
leading to reduced LC risk [71].
CYP2A13
CYP2A13, the primary CYP isoenzyme to activate
NNK, is highly expressed in the human respiratory
tract [72–74]. Wang et al. [75] showed that variant
genotypes of the Arg257Cys SNP of CYP2A13 were
associated with substantially decreased LC risk with
anOR of 0.41 (0.23–0.71). Furthermore, Zhang et al.
[76] reported that individuals homozygous for the
variant allele exhibited a 2-fold decrease in NNK ac-
tivation efﬁciency, and heterozygotes had 37–56%
lower metabolic activity.

Epidemiologic risk factors in LC 2

2009-03-30T15:00:00.000-07:00

Folate, isothiocyanates, and phytoestrogens
Folate deﬁciency is implicated in in vitro studies
in alterations in DNA methylation, DNA synthesis,
and disruption of DNA repair activities [45]; how-
ever, observational studies have yielded inconsis-
tent results [46–49]. In the New York State [46] and
Netherland Cohort Studies [48], an inverse associa-
tion between folate intake and LC riskwas reported.
Shen et al. [49] also reported that dietary folate in-
take was associated with a 40%reduction in LC risk
among former smokers. In contrast, data from the
Nurse’s Health Study revealed a lack of association
[47].
Isothiocyanates (ITCs) are nonnutrient com-
pounds in cruciferous vegetables with anticarcino-
genic properties. One possible mechanism for their
protective action is through downregulation of cy-
tochrome P-450 biotransformation enzyme levels
and induction of phase II enzymes [50,51]. ITCs can
also induce apoptosis, cell cycle arrest, and cell dif-
ferentiation [52]. Brennan et al. [53] showed that
weekly consumption of cruciferous vegetables pro-
tected against LC in those who were GSTM1 null
(OR = 0.67; 95% CI, 0.49–0.91), GSTT1 null (0.63,
0.37–1.07), or both (0.28, 0.11–0.67). Similar pro-
tective results were noted for consumption of spe-
ciﬁc cruciferous vegetables.However, Spitz et al. [54]
reported that current smokers with both low ITC
dietary intake and the GSTM1 null genotype or the
GSTT1 null genotype exhibited increased LC risk,
with ORs of 2.22 (1.20–4.10) and 3.19 (1.54–6.62),
respectively. This association was conﬁrmed by Gao
et al. [55]. The comparable OR in the presence of
both null genotypes was 5.45 (1.72–17.22). Results
in former smokers were not statistically signiﬁcant.
Dietary phytoestrogens are plant-derived nons-
teroidal compounds with weak estrogen-like activ-
ity. A signiﬁcant reductions in risk of LC with in-
creased phytoestrogen intake was observed [56].
The highest quartile of intake of total phytoestro-
gens from food sources was associated with a 46%
reduction in risk (OR = 0.54; 95% CI, 0.42–0.70;
p < 0.001 for trend). Several studies in Asian pop-
ulations, whose diet contains large quantities of
phytoestrogens, also reported reduced risk of LC
associated with high intakes of phytoestrogen
[57–61].

Epidemiologic risk factors in LC

2009-03-30T14:59:00.002-07:00

Smoking
Cigarette smoking
Cigarette smoke contains >80 carcinogens evalu-
ated by the International Agency for Research on
Lung Cancer, 3rd edition. Edited by Jack A. Roth, James D. Cox,
and Waun Ki Hong. c 2008 Blackwell Publishing,
ISBN: 978-1-4051-5112-2.
Cancer (IARC) [6] with “sufﬁcient evidence for
carcinogenicity” in humans or lab animals. While
smokers are at higher risk of LC than those who
have never smoked, there is substantial variation
in LC risk among smokers [7,8]. Peto et al. [7]
related UK national trends in smoking, smoking
cessation, and LC to the contrasting results from
two case–control studies of smoking and LC in the
United Kingdom[9]. Results showed large increases
in cumulative LC risk among continuing smokers
in 1990 data, reﬂecting prolonged smoking expo-
sure. Among bothmen and women in 1990, former
smokers had lower LC rates than continuing smok-
ers, with the reduction increasing substantially for
increased time since quitting. This study stresses the
importance of quitting smoking at an earlier age
to avoid subsequent risk of LC. The lifetime risk
of developing LC remains high for former smok-
ers, no matter how long the period of abstinence;
however, longer duration of abstinence is associated
with greater reductions in risk [7]. The relationship
between ETS exposure and LC has been extensively
evaluated in many epidemiologic studies. Results
from several meta-analyses report a positive asso-
ciation [10,11].
Family history
There have been a number of published studies
showing familial aggregation of LCs in ﬁrst-degree
relatives of probands with LC [12–18]. Schwartz
et al. [17] showed that the LC risk in a ﬁrst-degree
relative was associated with a 7.2-fold (95%
conﬁdence interval (CI), 1.3–39.7) increased risk
among nonsmokers with early age at onset (40–
59 year old group). The association between an in-
creased risk of LC among ﬁrst-degree relatives has
been conﬁrmed in other studies [19,20]. On the
other hand, Kreuzer et al. [21] reported no evi-
dence of familial risk. Familial aggregation only pro-
vides indirect evidence for the genetic inﬂuence,
and could be due to common genetic proﬁles among
the familymembers, shared smoking patterns, or by
a combination of both factors.
Prior respiratory diseases
LC risk may be modiﬁed by a prior history of res-
piratory diseases such as asthma, bronchitis, em-
physema, and hay fever. It has been reported
that there was a signiﬁcant protective effect in
the association between hay fever and LC (odds
ratio (OR) = 0.58; 95% CI, 0.48–0.70), and a
signiﬁcantly increased risk associated with prior
physician-diagnosed emphysema (OR = 2.87; 95%
CI, 2.20–3.76) [22]. A signiﬁcantly lower frequency
of hay fever was observed among patients with ma-
lignancies of lung, colon, bladder, and prostate as
compared to controls [23]. It was suggested that
the protective effects were attributed to enhanced
immune surveillance resulting from better detec-
tion and destruction of malignant cells [16,23–27];
also possibly, anti-inﬂammatory agents used to treat
hay fever might contribute to this protection. In
contrast, Talbot-Smith et al. [24] and Osann [16]
found no association between hay fever and LC
risk.
In another large case–control study comprised of
2854 cases and 3116 controls from seven different
European countries, a history of eczema was in-
versely associated with LC risk with an OR of 0.61
(0.5–0.8) [28]. In a meta-analysis, asthma was a
signiﬁcant risk factor for LC among never smok-
ers with a pooled risk ratio (RR) of 1.9 (1.4–2.5)
when adjusted for ETS exposure [29]. Currently,
there is no consensus on the role of prior respiratory
diseases, other than emphysema, in LC and the em-
pirical evidence, which is not entirely consistent,
has been largely derived from observational epi-
demiologic data.
Environmental and occupational
exposures
Asbestos, arsenic, bischloromethylether,chromium,
nickel, polycyclic aromatic compounds, radon, and
vinyl chloride have all been implicated in LC etiol-
ogy, and have been reviewed extensively before.
Nutrition and dietary patterns
Fruits and vegetables
Observational studies strongly suggest that in-
creased vegetable and fruit intake is associated
with reduced risk of LC [30–33]. In the European
Prospective Investigation into Cancer and Nutrition
(EPIC), with data collected from 478,021 subjects, a
signiﬁcant inverse association between LC risk and
fruit consumption was observed after adjusting for
smoking and other confounders (RR = 0.60; 95%
CI, 0.46–0.98 for the highest quintile compared to
the lowest); however, there was no such associa-
tion with vegetable consumption [33]. In a large
prospective Danish cohort study comprising 54,158
participants, the incidence rate of LC was highest
in the lowest quartile of plant food intake (fruit,
vegetable, legumes, and potatoes) [34]. Neuhouser
et al. [35] in a pooled analysis of eight prospective
studies with a total of 3206 incident LC cases oc-
curring among 430,281 individuals followed for 6–
16 years reported that compared to the lowest quin-
tile of consumption, the RRs of the highest quintile
consumption for total fruits, total fruits and vegeta-
bles, and total vegetables were 0.77 (0.67–0.87; p <
0.001), 0.7 (0.69–0.90; p = 0.001), and 0.88 (0.78–
1.00; p = 0.12), respectively. They concluded that
elevated fruit and vegetable consumption, mostly
due to fruit intake, is associated with a modest re-
duction in LC risk [35]. Overall, the association
between high intake of fruit and vegetables and re-
duced LC risk appears conclusive but what subtypes
of fruits and vegetables and which micronutrients
contribute to this protection remain controversial.
Carotenoids
Carotenoids are red and yellow fat-soluble pig-
ments found in many fruits and vegetables.
Numerous studies have shown that a diet high
in total carotenoids is protectives, but results are
inconsistent in the association between individual
carotenoids and LC. In a pooled analysis of seven
cohort studies in North America and Europe based
on a follow-up of 7–16 years with 3155 incident LC
cases among 399,765 participants, Mannisto et al.
[36] reported that only β-cryptoxanthin intake
was inversely associated with LC risk with a RR of
0.76 (0.67–0.86) even after controlling for intake of
vitamin C, folate, other carotenoids, multivitamin
use, and smoking status. In the Alpha-Tocopherol,
β-carotene Cancer Prevention Study (ATBC) [37],
with 1644 incident LC cases, during 14 years
of follow-up, lower risks were observed for the
highest versus the lowest quintiles of lycopene
(28% reduction), lutein/zeaxanthin (17%), β-
cryptoxanthin (15%), total carotenoids (16%),
serumβ-carotene (19%), and serumretinol (27%),
while intakes of β-carotene, α-carotene, and retinol
were not associated with signiﬁcant reduction. In
a pooled analysis of the Nurse’s Health Study and
the Health Professional Follow-up Study (HPFS),
Michaud et al. [38] reported that only α-carotene
and lycopene intakes were signiﬁcantly associated
with lower risk of LC. In overall analyses of all
carotenoids combined, LC risk was signiﬁcantly
lower in subjects with high total carotenoid intake
with RR of 0.68 (0.49–0.94). Inadequate adjust-
ment for confounding, especially smoking factors,
and the lack of consideration of multicollinearity
between individual carotenoids may be responsible
for inconsistent results across studies.
Data generated by the Beta-Carotene and Retinol
Efﬁcacy Trial (CARET) and ATBC trials failed to con-
ﬁrm the protective role of β-carotene in smokers
[35,37]. Contrary to the expectation and observa-
tional epidemiologic evidence, supplementation of
β-carotene resulted in a surprisingly increased over-
all LC incidence and higher total mortality among
current smokers [39–41]. Debate has been focused
on dosage, duration of trials, and the difference
between dietary intake and supplement use [42].
Preclinical data provide biologic plausibility for this
adverse interaction between cigarette smoking and
β-carotene [43]. Recently, in a cohort of French
Women [44], high intake of β-carotene was signiﬁ-
cantly protective against LC among never smokers,
but was associated with increased LC incidence in
ever smokers. Tests for interaction between smok-
ing and β-carotene intake were signiﬁcant [44].

CHAPTER 3 Lung Cancer Susceptibility and Risk Assessment Models

2009-03-30T14:59:00.001-07:00

Introduction
In 2007, it is estimated that there will be 213,380
new cases of lung cancer (LC) and 160,390 LC-
related deaths in the United States [1]. These deaths
represent 31% of total mortality from all cancers in
US men and 26% in women. While tobacco smok-
ing is the predominant cause of LC, a variety of
other exposures, such as family history of LC, var-
ious chronic respiratory diseases, and environmen-
tal tobacco smoke (ETS), are also linked to elevated
LC risk. Host susceptibility may also be involved in
LC risk, since only a fraction of smokers develops LC
[2–5]. Because carcinogenesis is amultistep process,
multiple molecular events during this process ac-
count for the malignant transformation upon initial
carcinogenic exposure. Understanding that multi-
ple components contributing to LC can lead to the
identiﬁcation of high-risk subgroups,whomay ben-
eﬁt from targeted screening or other interventions.
Here, we provide a summary of recent advances in
the molecular epidemiology of LC.

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2009-03-30T14:58:00.001-07:00

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Association of common alleles of small effect (polymorphisms) with lung cancer risk

2009-03-30T14:57:00.000-07:00

Results of hundreds of studies using association
analysis to evaluate the effects of various polymor-
phisms, in metabolic genes, growth factors, growth
factor receptors, markers of DNA damage and re-
pair and genomic instability, and in oncogenes and
tumor suppressor loci have been published. Many
of these studies have yielded inconsistent results.
The effects of risk alleles at these loci are expected
to be individually small, and they may interact with
smoking and/or other loci to increase lung cancer
risk. These association studies are beyond the scope
of this chapter. Two recent reviews [100,101] can
help the reader obtain an overview of these studies.
Discussion
All these lines of evidence suggest that there may
be one or several genes causing inherited increased
risk to lung cancer in the general population.While
association studies have given evidence that alle-
les at various genetic loci may inﬂuence lung can-
cer risk, there has frequently been disagreement be-
tween studies. The ﬁrst linkage study of lung cancer
has given signiﬁcant evidence of linkage to a region
on chromosome 6q. If a susceptibility locus is iden-
tiﬁed in this region, it will be of major public health
importance as it will allow identiﬁcation of individ-
uals at especially high risk who can then be targeted
for intensive efforts at environmental risk reduc-
tion. In addition, identiﬁcation of such a gene will
lead to better understanding of the mechanism of
carcinogenesis in general, perhaps eventually lead-
ing to better methods of prevention and treatment.
Conﬁrmation of a genetic predisposition for lung
cancer can be obtained by ﬁnding evidence for link-
age of the putative susceptibility gene(s) to genetic
marker loci in a speciﬁc chromosomal region(s).
One potential problem in the search for such a link-
age is heterogeneity. There are different types of
heterogeneity of this disease and of its etiological
factors: (1) there is heterogeneity at the level of
histological types of lung cancer, (2) there is het-
erogeneity at the level of exposure to a variety of
environmental risk factors, and (3) there could be
heterogeneity at the level of inherited susceptibil-
ity loci, i.e., there could be one locus involved in
susceptibility for one family and a different locus
involved in susceptibility for another family. All of
these types of heterogeneity could possibly con-
found the identiﬁcation of a susceptibility locus (or
loci) for lung cancer. The suggestive evidence in the
published linkage study [99] for susceptibility loci
at several other regions of the genome supports the
possibility of locus heterogeneity in lung cancer.
If, through linkage and positional cloning tech-
niques, a genetic locus or loci that contributes to
inheritable risk for lung cancer can be identiﬁed, or
one of the candidate loci suggested to modify risk
by association studies can be conﬁrmed as a sus-
ceptibility locus, then the effects of the alleles at
this locus and its interaction with cigarette smok-
ing and the other well-known environmental risk
factors for lung cancer can be elucidated with much
more accuracy than presently possible and our un-
derstanding of lung carcinogenesis in general may
be increased.

Linkage analysis of lung cancer

2009-03-30T14:56:00.000-07:00

Linkage analysis is a statistical analysis of pedi-
gree data that looks for evidence of cosegregation
through the generations in human pedigrees of al-
leles at a genetic “susceptibility” locus and some
known genetic “marker” locus (usually a DNA poly-
morphism). Linkage analysis is a very powerful
method for detecting genetic loci that are highly
penetrant (after adjusting for environmental risk
factors). However, power decreases as the suscep-
tibility allele becomes more common and less pene-
trant. Since cigarette smoking is an extremely strong
risk factor for lung cancer (e.g., 4), it is important
that one looks for a major gene after controlling for
at least personal smoking, as this will increase the
power to detect linkage.
Bailey-Wilson et al. [99] published the ﬁrst ev-
idence of linkage of a putative lung cancer sus-
ceptibility locus to a region of chromosome 6q.
Data were collected at eight recruitment sites of the
Genetic Epidemiology of Lung Cancer Consortium
(GELCC): the University of Cincinnati, University of
Colorado, Johns Hopkins School of Public Health,
Karmanos Cancer Institute, Saccomanno Research
Institute, Louisiana State University Health Sciences
Center, Mayo Clinic, and Medical College of Ohio.
Of the 26,108 lung cancer cases screened at GELCC
sites for this study, 13.7% had at least one ﬁrst-
degree relative with lung cancer. Following the ini-
tial family history screening process, we collected
additional information from the 3541 families with
at least one ﬁrst-degree relative with lung cancer.
We interviewed probands and/or their family rep-
resentatives to collect data regarding additional per-
sons affected with any cancers in the extended fam-
ily, vital status of affected individuals, availability of
archival tissue, and willingness of family members
to participate in the study. Further pedigree devel-
opment and biospecimen collection (blood, buccal
cells, or ﬁxed tissue) were performed on 771 fami-
lieswith three ormore ﬁrst-degree relatives affected
with lung cancer. Cancers were veriﬁed by medical
records, pathology reports, cancer registry records,
or death certiﬁcates for 69% of individuals affected
with either lung or throat cancer (LT), and by reports
of multiple family members for the other 31% of
family members affected with LT. Of these families,
only 52 had enough biospecimens available tomake
theminformative for linkage analyses. DNA isolated
from blood was genotyped at the Center for Inher-
ited Disease Research (CIDR, a National Institutes of
Health-supported core research facility), and DNA
from buccal cells and archival tissue and sputum
were genotyped at the University of Cincinnati, for
a panel of 392 microsatellite (short tandem repeat
polymorphism, STRP) marker loci. The data were
checked for errors and then analyzed using para-
metric and nonparametric linkagemethods.Marker
allele frequencies were calculated separately and
linkage analyses were performed separately for the
white American and African American families,
with the results combined in overall tests of linkage.
Our primary analytical approach assumed a
modelwith 10%penetrance in carriers and 1%pen-
etrance in the noncarriers. This analytical approach
weights information only fromthe affected subjects.
For this analysis we used FASTLINK for two-point
analysis and SIMWALK2 formultipoint analysis.We
chose this linkage model as our primary analytical
approach because of uncertainty about the strength
of relationship between smoking behavior and lung
cancer risk in the high-risk families we are study-
ing, and because the complex “gene + environ-
ment” models from the published segregation anal-
yses were not currently available in any multipoint
linkage analysis program. In addition, since about
90% of the affected family members in our studies
smoked, weighting only the affected individuals in
our simple dominant, lowpenetrancemodel has the
effect of jointly allowing for smoking status, while
ignoring information from unaffected subjects. We
allowed for genetic heterogeneity (different families
having different genetic causation) in the analysis.
Secondary analyses usedmore complexmodels that
included age and pack-years of cigarette smoking to
modify the penetrances. Our standard for this anal-
ysis was LODLINK, which uses the genetic regres-
sive model, obtained from segregation analyses by
Sellers et al. [80] and Bailey-Wilson et al. [91]. The
current implementation of LODLINK only permit-
ted two-point analysis when a covariate is included
and it is well known that two-point linkage is less
powerful in general than multipoint linkage analy-
sis. Nonparametric analyses were also performed as
secondary analyseswith variance componentsmod-
els using SOLAR (binary trait option) and mixed ef-
fects Cox regression models, in which time to onset
of disease is modeled as a quantitative trait.
Multipoint parametric linkage under the sim-
ple dominant low-penetrance affected-only model
(Figure 2.1) yielded amaximumheterogeneity LOD
score (HLOD) of 2.79 at 155 cM (marker D6S2436)
on chromosome 6q23–25 in the 52 families, with
67% of families estimated to be linked. Multipoint
analysis of a subset of 38 families with four affected
relatives gave an HLOD of 3.47 at this same loca-
tion, with 78% of families estimated to be linked,
whereas for the 23 highest risk families (ﬁve ormore
affected in two ormore generations), themultipoint
HLOD score was 4.26, with 94% of these families
estimated to be linked to this region. Our non-
parametric analyses and the two-point parametric
analyses that used the Sellers et al. model [80,91]
all provided additional support for linkage to this
region.
Additional families are nowbeing collected by the
GELCC to attempt to conﬁrm this linkage result in
an independent sample and to narrow the critical
region that may contain a susceptibility gene. In ad-
dition, several other regions showed suggestive ev-
idence of linkage and these are being pursued.

Oncogenes and tumor suppressor genes

2009-03-30T14:55:00.000-07:00

In addition to epidemiological evidence, experimen-
tal evidence of the role of genes in lung cancer cau-
sation has been accumulating. First, it seems prob-
able that genetic changes are responsible for the
pathogenesis of most, if not all, human malignan-
cies [95]. In particular, lung carcinogenesis is the re-
sult of a series of genetic mutations that accumulate
progressively in the bronchial epithelium, ﬁrst gen-
erating histologically identiﬁable premalignant le-
sions and ﬁnally resulting in an invasive carcinoma.
The premalignant genetic changes may occur many
years before the appearance of invasive carcinoma.
Cytogenetic and molecular studies have shown
that mutations in protooncogenes and tumor sup-
pressor genes (TSGs) are critical in themultistep de-
velopment and progression of lung tumors. Allele
loss analyses have implicated the presence of other
tumor suppressor genes involved in lung tumorige-
nesis. These studies revealed frequent occurrences
of chromosomal deletions including regions of 3p,
5q, 8p, 9p, 9q, 11p, 11q, and 17q. These studies are
outside the scope of this chapter (see e.g. [96–98]
for reviews).

Weaknesses of familial aggregation and segregation analyses

2009-03-30T14:53:00.000-07:00

While most of these studies included measures of
personal smoking on the cases (or probands) and
controls in the models, some of the aggregation
studies did not include measures of amount of
cigarette smoking in the relatives and only one in-
cluded measures of passive smoking. The segrega-
tion analyses did not include passive smoking or oc-
cupational risk factors in the models, and only one
of these three studies collected data on history of
chronic bronchitis. Furthermore, segregation anal-
yses are not sufﬁcient to prove the existence of a
major locus because only a subset of all possible
models can be tested. These segregation analyses
did not, for example, model the large number of
possible oligogenic cases where two or three major
loci act in conjunction with smoking to affect risk of
lung cancer.

Segregation analyses of lung- and smoking-associated cancers

2009-03-30T14:51:00.000-07:00

Given the evidence for familial aggregation of lung
and other smoking-associated cancers, after ac-
counting for personal tobacco use and occupa-
tional/industrial risk factors, segregation analyses
have been performed to determine whether pat-
terns of transmission consistent with at least one
major, high-penetrance genetic locus may be in-
volved in lung cancer risk.
Sellers et al. [80] performed genetic segregation
analyses on the lung cancer proband families of Ooi
et al. [54] described above. The trait was expressed
as a dichotomy, affected or unaffected with lung
cancer. The analyses used the general transmission
probability model [81], which allows for variable
age of onset of the lung cancer [82–84]. The likeli-
hood of themodelswas calculated using a correction
factor appropriate for single ascertainment [85,86],
i.e., conditioning the likelihood of each pedigree on
the probands being affected by their ages at exami-
nation or death.
Age of onset of lung cancer was assumed to fol-
low a logistic distribution that depended on pack-
years of cigarette consumption and its square, an
age coefﬁcient and a baseline parameter. Results in-
dicated compatibility of the data with Mendelian
codominant inheritance of a rare major autosomal
gene that produces earlier age of onset of the can-
cer. Segregation at this putative locus could account
for 69 and 47%of the cumulative incidence of lung
cancer in individuals up to ages 50 and 60, respec-
tively. The genewas predicted to be involved in only
22% of all lung cancers in persons up to age 70, a
reﬂection of an increasing proportion of noncarriers
succumbing to the effects of long-term exposure to
tobacco [80,87].
Additional segregation analysis of these families
was performed to determine whether evidence ex-
ists for a major gene that increases susceptibility to
a group of smoking-associated cancers rather than
just lung cancer alone. The trait was deﬁned as
unaffected or affected with cancer at any of the
following sites: lung, lip, oral cavity, esophagus, na-
sopharynx, trachea, bronchus, larynx, cervix, blad-
der, kidney, colon/rectum, and pancreas. The results
were compatible with segregation of a major gene
that inﬂuences age-of-onset of cancer. The hypothe-
ses ofMendelian dominant, codominant, and reces-
sive inheritance could not be rejected but, according
to Akaike’s Information Criterion [88], Mendelian
codominant inheritance provided the best ﬁt to
the data [89]. Additional analyses suggest that bet-
ter ﬁt of Mendelian inheritance of an allele that
acts with smoking to inﬂuence the risk of can-
cer is obtained if a somewhat different cluster of
smoking-associated cancers is considered “affected”:
lung, oral cavity, esophagus, nasopharynx, lar-
ynx, pancreas, bladder, kidney, and uterine cervix
[90].
Segregation analyses of these data ([91] and
our unpublished results) using Class A regressive
models showed signiﬁcant evidence for a poly-
genic/multifactorial component in addition to the
major gene component described above. Inclusion
of this polygenic/multifactorial component signiﬁ-
cantly improved the ﬁt of the model to the data
without changing the basic conclusions of the pre-
vious analyses, i.e., that evidence exists for a major
locus with a codominant susceptibility allele that
acts in conjunction with smoking, and that all mod-
els excluding such amajor gene effectwere rejected.
Gauderman et al. [92] reanalyzed these same data
using a Gibbs sampler method to examine gene by
environment interactions and found evidence for a
major dominant susceptibility locus that acts in con-
junctionwith cigarette smoking to increase risk; this
model was very similar to the previous results since
the codominant Mendelian models predicted very
small numbers of homozygous susceptibility allele
carriers.
Yang and her coworkers performed complex seg-
regation analysis on the families of nonsmoking
lung cancer probands in metropolitan Detroit [93].
Evidence was found for Mendelian codominant in-
heritance with modifying effects of smoking and
chronic bronchitis in families of nonsmoking cases
diagnosed at ages 40–59. The estimated risk allele
frequency was 0.004. While homozygous individu-
als with the risk allele are rare in the study popula-
tion, penetrance was very high for early-onset lung
cancer (85% in males and 74% in females by age
60). The probability of developing lung cancer by
age 60 in individuals heterozygous for the rare al-
lele was low in the absence of smoking and chronic
bronchitis (7% in males and 4% in females) but in
the presence of these risk factors it increased to 85%
in males and 74% in females, which was the same
level predicted for homozygotes. The attributable
risk associatedwith the high-risk allele declineswith
age, when the role of tobacco smoking and chronic
bronchitis become more important.
Wu et al. [94] performed complex segregation
analysis of families of 125 female, nonsmoking
lung cancer probands in Taiwan. These lung cancer
probandswere diagnosedwith lung cancer between
1992 and 2002 at two hospitals in Taiwan. Complete
data on patients, spouses and ﬁrst-degree relatives
were collected for 108 families.Data collected on the
patients and their relatives included demographic,
life-style, and medical history variables. Complex
segregation analysis using logistic models for age at
onset, including pack-years of cigarette smoking in
the model was performed on 58 of these families.
An ascertainment correction was made using the
phenotype of the probands, but this may have been
inadequate since the 58 families were a subset of
the 108 families where there was at least one addi-
tional affected relative in the family. TheMendelian
codominant model that included risk due to per-
sonal smoking ﬁt the data best, signiﬁcantly better
than the sporadic or purely environmental mod-
els. This model was not rejected against the general
model in an early-onset (less than 60 years) subset
of the families but was rejected in the later-onset
families and the total dataset.
Taken together, the Taiwan, Detroit, and
Louisiana studies share remarkably similar results
and demonstrate statistical evidence for at least one
major gene that acts in conjunction with personal
smoking and possibly chronic bronchitis to increase
risk of lung cancer.

2009-03-30T14:49:00.000-07:00

history of neoplastic disease exclusive of the
proband (62%of patients’ families and 57%of con-
trol families). However, the families of the lung can-
cer cases were more likely (30%) to have two or
more familymembers affectedwith any cancer than
the families of the controls. The case families were
also signiﬁcantly more likely to have two or more
relatives with lung cancer than were the control
families. In addition, a higher percentage of all pri-
mary tumors were lung tumors (16.5%) in patients’
relatives as compared to controls’ relatives (10%).
The progression of increased risks for observing 1,
2, 3, and 4+ affected relatives in case families ver-
sus control families was slightly smaller than in the
Sellers et al. study [55] but showed the same type of
progression.
Family history data froman incident case–control
study in Texaswere analyzed for evidence of familial
aggregation by Shaw et al. [57]. A total of 943 histo-
logically conﬁrmed lung cancer cases and 955 age-,
gender-, vital-status-, and ethnicity-matched con-
trols were interviewed regarding smoking, alcohol
use, cancer in ﬁrst-degree relatives, medical history,
and demographic characteristics. After adjusting for
personal smoking status, passive smoking exposure
(ever/never), and gender, participants with at least
one ﬁrst-degree relativewith lung cancer had a lung
cancer risk of 1.8 compared to those with no rela-
tives with lung cancer. Lung cancer risk increased as
the number of relatives with cancer increased and
was highest when only relatives with lung cancer
were considered (odds ratios of 1.7 and 2.8 for one
and two or more relatives with lung cancer, respec-
tively). Lung cancer was diagnosed at a signiﬁcantly
younger age among cases who had ﬁrst-degree rel-
atives with lung cancer than among those who had
no relatives with lung cancer. However, no such age
difference was seen between cases who had ﬁrst-
degree relatives with any cancer versus those who
had no relatives with cancer. This study also exam-
ined histologic subtypes of lung cancer cases and
found that for each histologic type, there were sig-
niﬁcant risks associated with having any relatives
with lung cancer, with odds ratios of 2.1 for ade-
nocarcinoma, 1.9 for squamous cell carcinoma, and
1.7 for small cell lung cancer. Finally, in this study,
only current and former smokers had an increased
lung cancer risk associated with lung cancer in
relatives.
Cannon-Albright et al. [58] examined the degree
of relatedness of all pairs of lung cancer patients in
the Utah Population Database. By comparing this to
the degree of relatedness in sets ofmatched controls,
they showed that lung cancer exhibited excess fa-
miliality, and three of four histological tumor types
still showed excess familiality when considered sep-
arately. In the same population, but using different
methodology, Goldgar et al. [59] studied lung can-
cer probands and controls who had died in Utah
and their ﬁrst-degree relatives. They found that 2.55
timesmore lung cancers occurred in ﬁrst-degree rel-
atives of lung cancer probands than expected based
on rates in control relatives. When they stratiﬁed
by gender, they observed higher relative risks for
female relatives of female probands (FRR = 4.02)
versusmale relatives ofmale probands (FRR = 2.5).
No adjustment was made in these analyses for per-
sonal smoking or other environmental risk factors,
so these results may simply reﬂect the familiality of
smoking behaviors. However, this is a largely non-
smoking population and Utah has the lowest smok-
ing rates of any state in the United States.
The number of lung cancers observed in some
twin studies have been too small to draw con-
clusions regarding familiality of lung cancer [60]
although possible aggregation of bronchoalveolar
carcinoma has been suggested in twin and family
studies [61,62]. However, this effect may be due to
aggregation of cigarette smoking as risk of this can-
cer is linked to tobacco consumption [63]. In 1995,
a study using a large twin registry, the National
Academy of Sciences—National Research Council
Twin Registry, Braun et al. [64] reported that the
observed concordance rates of monozygotic (MZ)
twins for death from lung cancer compared to that
of dizygotic (DZ) twins was 1.1 (95% conﬁdence
interval, 0.6–1.9) although this did not adjust for
smoking behaviors in the twins. These results sug-
gest that, as expected, on a population level, smok-
ing behavior is probably a much stronger risk factor
than inherited genetic susceptibility.
Studies of familial risk of lung cancer in non-
smokers [65–67] have also shown increased risk
of lung cancer associated with a family history of
lung cancer. The study by Schwartz et al. [65] found
increased risk of lung cancer among relatives of
younger, nonsmoking lung cancer cases as com-
paredwith relatives of younger controls after adjust-
ing for smoking, occupational and medical histories
of each family member, suggesting increased sus-
ceptibility to lung cancer among relatives of early-
onset nonsmoking lung cancer patients. Wu et al.
[66] found an increased risk of lung cancer in per-
sonswith a history of lung or aerodigestive tract can-
cer in ﬁrst-degree relatives after adjustment for ETS
exposure, which was signiﬁcant for affected moth-
ers and sisters. Mayne et al. [67], in a population-
based study of nonsmokers (45% never smokers
and 55% former smokers who had quit at least
10 years prior to diagnosis or interview; 437 lung
cancer cases and 437 matched population controls)
in New York State, found that after adjusting for age
and smoking status (yes, no) in the relatives, a posi-
tive history in ﬁrst-degree relatives of any cancer or
lung cancer or aerodigestive tract cancer or breast
cancer were each associated with signiﬁcantly in-
creased risk of lung cancer.
In 2000, Bromen et al. [68], in a population-based
case–control study in Germany, showed that lung
cancer in parents or siblings was signiﬁcantly as-
sociated with an increased risk of lung cancer and
that this risk was much stronger in younger partic-
ipants. In 2003, Etzel et al. [69] evaluated whether
ﬁrst-degree relatives of lung cancer caseswere at in-
creased risk for lung cancer and for other smoking-
related cancers (bladder, head and neck, kidney, and
pancreas). They studied 806 hospital-based lung
cancer patients and 663 controls matched on age,
sex, ethnicity, and smoking history, all from the
Houston, Texas, area. After adjustment for smok-
ing history of patients and their relatives, there
was signiﬁcant evidence for familial aggregation of
lung cancer and of smoking-related cancers. How-
ever, they did not ﬁnd increased aggregation in
the families of young onset (less than or equal to
age 55) lung cancer cases or in families of never-
smokers.
Two studies in China [70,71] found, after adjust-
ing for age, sex, birth order, residence, family size,
chronic obstructive pulmonary disease (COPD),
smoking and cumulative index of smoky coal ex-
posure or occupational/industrial exposure index,
that ﬁrst-degree relatives of lung cancer patients
were at signiﬁcantly increased risk for lung cancer
compared to the same relatives of controls. They
also observed that families of the lung cancer pa-
tientswere signiﬁcantlymore likely to have three or
more affected relatives than were control families.
A series of studies using the Swedish Family-
Cancer Database [72–75], which totals over
10.2 million individuals, found that a high propor-
tion of lung cancers diagnosed before the age of 50
appear to be heritable, and that lung cancer patients
with a family history of lung cancer were at a sig-
niﬁcantly increased risk of subsequent primary lung
cancers.
In the United Kingdom, a case–control study of
lung cancer prevalence in ﬁrst-degree relatives of
1482 female lung cancer cases and 1079 female
controls [76] was performed, adjusting for age and
tobacco exposure (pack-years) in the cases and
controls. They found that lung cancer in any ﬁrst-
degree relative was associated with a signiﬁcant in-
crease in lung cancer risk, and that the increase in
risk was stronger in relatives of cases with onset less
than 60 years or cases with three or more affected
relatives. However, this study was not able to adjust
for personal smoking in the relatives since these data
were not available.
A study ofwhite and black relatives of early-onset
lung cancer cases and of 773 frequency-matched
controls in Detroit, Michigan [77], showed that
smokers with a family history of early-onset lung
cancer had a higher risk of lung cancerwith increas-
ing age than smokers without a family history, and
that relatives of black cases were at higher risk than
relatives of white cases, after adjusting for age, sex,
pack-years of cigarette smoking, pneumonia, and
COPD.
A recent study [78] utilizing the Icelandic Can-
cer Registry calculated risk ratios of lung cancer in
ﬁrst-, second-, and third-degree relatives of 2756
lung cancer patients diagnosed between 1955 and
2002. Relative risks were signiﬁcantly elevated for
all three classes of relatives, and this increased risk
was stronger in relatives of early-onset lung cancer
patients (age at onset less than or equal to 60 years).
The effect did not appear to be solely due to the
effects of smoking in all relative types, except for
cousins and spouses.
A review in 2005 by Matakidou et al. [79] of 28
case–control, 17 cohort and 7 twin studies of the re-
lationship between family history and risk of lung
cancer and a meta-analysis of risk estimates, con-
cluded that the case–control and cohort studies con-
sistently showan increased risk of lung cancer given
a family history of lung cancer, and that risk appears
to be increased given a history of early-onset lung
cancer or of multiple affected relatives. However,
the results of the twin studies and the observed in-
creased risk of disease in spouses highlighted the
importance of environmental risk factors, such as
smoking, in this disease.

Evidence for familial aggregation of lung cancer

2009-03-30T14:48:00.001-07:00

Tokuhata and Lilienfeld [48,49] provided epidemi-
ologic evidence for familial aggregation of lung can-
cer over 40 years ago. After accounting for personal
smoking, their results suggested the possible inter-
action of genes, shared environment, and common
life-style factors in the etiology of lung cancer. In
their study of 270 lung cancer patients and 270 age-,
sex-, race-, and location-matched controls and their
relatives, they found a relative risk of 2–2.5 formor-
tality due to lung cancer in cigarette-smoking rel-
atives of cases as compared to smoking relatives
of controls. Nonsmoking relatives of lung cancer
cases were also at higher risk when compared to
nonsmoking relatives of controls. Smoking was a
more important risk factor for males but family his-
tory was the more important risk factor for females.
They also noted a synergistic interaction between
familial and smoking factors on the risk of lung
cancer in relatives, with smoking relatives of lung
cancer patients having much higher risk of lung
cancer than either nonsmoking relatives of patients
or smoking relatives of controls. They observed a
substantial increase in mortality due to noncancer-
ous respiratory diseases in relatives of patients as
compared to relatives of controls, suggesting that
the case relatives have a common susceptibility to
respiratory diseases. However, they found no signif-
icant differences between the spouses of the lung
cancer cases and controls for lung cancer mortality,
mortality from noncancerous respiratory diseases,
or smoking habits.
The major weakness of this study was that smok-
ing status alonewas used, as nomeasures of amount
or duration of smoking in the relatives were avail-
able. Therefore, some of the familial aggregation
could be due to familial correlation in smoking lev-
els or age at starting smoking. However, nonsmok-
ing relatives of cases were at higher risk than non-
smoking relatives of controls.
Since this time, many other studies have shown
evidence of familial aggregation of lung cancer. In
1975, Fraumeni et al. [50] reported an increased
risk of lung cancer mortality in siblings of lung can-
cer probands. In 1982, Goffman et al. [51] reported
families with excess lung cancer of diverse histo-
logic types. Lynch et al. [52] reported evidence for
increased risk of cancer at all anatomic sites for rel-
atives of lung cancer patients but no signiﬁcant in-
creased risk for lung cancer alone in these relatives.
Leonard et al. [53] reported that survivors of famil-
ial retinoblastoma may also be at increased risk for
small cell lung cancer.
In southern Louisiana, our retrospective case–
control studies reported an increased familial risk for
lung cancer [54] and nonlung cancers [55] among
relatives of lung cancer probands after allowing for
the effects of age, sex, occupation, and smoking.
In these two studies, familial aggregation analy-
ses were performed on a set of 337 lung cancer
probands (cases), their spouse controls, and the par-
ents, siblings, half-siblings, and offspring of both
the probands and the controls. The probands were
male and female Caucasians who died from lung
cancer during the period 1976–1979 in a 10-parish
(county) area of southern Louisiana, a region noted
for its high lung cancer mortality rates. There were
about 3.5 male probands to every female lung can-
cer proband in the dataset. A strong excess risk for
lung cancer was detected among ﬁrst-degree rela-
tives of probands compared to relatives of spouse
controls, after adjusting for age, sex, smoking status,
total duration of smoking, cigarette pack-years, and
a cumulative index of occupational/industrial ex-
posures. Parents of probands had a fourfold risk of
having developed lung cancer as opposed to parents
of spouses, after adjusting for the effects of age, sex,
smoking, and occupational exposures. Females over
40 years old who were relatives of probands were
at nine times higher risk than similar female rela-
tives of spouses, even among nonsmokers who had
not reported excessive exposure to hazardous occu-
pations. Among female heavy smokers who were
relatives of probands, the risk was increased four-
to sixfold. Overall, male relatives of probands had a
greater risk of lung cancer than their female coun-
terparts. After controlling for the confounding ef-
fects of the measured environmental risk factors,
relationship to a proband remained a signiﬁcant de-
terminant of lung cancer, with a 2.4 odds in favor
of relatives of probands.
These same families were reanalyzed [55] to de-
termine if nonlung cancers exhibited similar familial
aggregation.When analyzing the number of cancers
at any site that occurred in a family, proband fam-
ilies were found to be 1.67 times more likely than
spouse families to have one family member (other
than the proband)with cancer, and 2.16 timesmore
likely to have two family members with cancer. For
three cancers and four or more cancers, the relative
risk increased to 3.66 and 5.04, respectively. Each
risk estimate was signiﬁcant at the 0.01 level. The
most striking differences in cancer prevalence be-
tween proband and control families were noted for
cancer of the nasal cavity/sinus, mid-ear, and lar-
ynx (odds ratio, OR = 4.6); trachea, bronchus and
lung (OR = 3.0); skin (OR = 2.8); and uterus, pla-
centa, ovary, and other female organs (OR = 2.1).
After controlling for age, sex, cigarette smoking, and
occupational/industrial exposures, relatives of lung
cancer probands maintained an increased risk of
nonlung cancer (p < 0.05) when compared to rel-
atives of spouse controls.
A family case–control study, drawn from
a population-based registry in Saskatchewan,
Canada, was reported by McDufﬁe [56]. A total
of 359 cases and 234 age- and gender-matched
community controls were included in the study.
Most families reported at least one member with a

Biologic risk factors

2009-03-30T14:46:00.002-07:00

In general, all studies suggesting genetic suscepti-
bility have also shown strong risk due to cigarette
smoking and often have shown an interaction of
high-risk genotype and smoking on lung cancer
risk. When trying to determine whether a complex
disease or trait such as lung cancer has a genetic
susceptibility, one asks three major questions:
1 Does the disease (lung cancer) cluster in families?
If some risk for lung cancer is inherited, then one
would expect to see clustering of that trait in some
families above what would be expected by chance.
2 If the aggregation of lung cancer does occur in
some families, can the observation be explained by
shared environmental/cultural risk factors? In this
disease, one needs to assess whether the familial
clustering of lung cancer is solely due to clustering
of smoking behaviors or other environmental expo-
sures within families.
3 If the excess clustering in families is not explained
by measured environmental risk factors, is the pat-
tern of disease consistent with Mendelian transmis-
sion of a major gene (i.e., of transmission through
some families of a moderately high penetrance risk
allele) and can this gene(s) be localized and identi-
ﬁed in the human genome.

Inhalation of tobacco smoke

2009-03-30T14:46:00.001-07:00

The association between cigarette smoking and lung
cancer is strong and well established (e.g. [3,5,6,37–
42]). The incidence of lung cancer is correlated with
the cumulative amount and duration of cigarettes
smoked in a dose–response relationship [7,38,43]
and smoking cessation results in decreased risk of
the disease, with the amount of decrease being re-
lated to time elapsed since the individual stopped
smoking [7,44]. Lung cancer rates and smoking
rates are also highly correlated in different geo-
graphic regions [45]. In 1991, Shopland et al. [46]
showed that the relative risk of lung cancer formale
smokers versus nonsmokers is 22.36 and that for fe-
male smokers versus female nonsmokers is 11.94.
They also estimated that 90% of lung cancers in
men and 78% in women were directly attributable
to tobacco smoking. Kondo et al. [47] showed a
signiﬁcant (p < 0.001) dose–response relationship
between number of cigarettes smoked and the fre-
quency of p53 mutations in tumors of lung cancer
patients, suggesting that somatic p53mutationsmay
be caused in some way by exposure to a carcino-
gen/mutagen in tobacco smoke or itsmetabolites. A
review by Anberg and Samet [30] discusses this evi-
dence of the role of cigarette smoking in lung cancer
causation inmore detail. None of the evidence given
below for genetic susceptibility loci should be con-
strued as suggesting that cigarette smoking is not
the main cause of lung cancer.

CHAPTER 2 Lung Cancer Susceptibility Genes

2009-03-30T14:45:00.000-07:00

Introduction
After heart disease, cancer is the most common
cause of death and lung cancer is the most common
cause of cancer death in the United States [1]. From
1950 to 1988, lung cancer experienced the largest
increase in mortality rate of all the cancers and lung
cancer caused an estimated 146,000 deaths in the
United States in 1992 [2]. Lung cancer became the
leading cause of cancer death in men in the early
1950s and in women in 1987.
Cancer of the lung has frequently been cited as an
example of a malignancy that is solely determined
by the environment [3,4] and the risks associated
with cigarette smoking [3–7] and certain occupa-
tions, such as mining [8], asbestos exposure, ship-
building, and petroleum reﬁning [9–14], are well
established. Most lung cancers are attributable to
cigarette smoking (e.g. [15]). Dietary studies have
found reduction in risk associated with high com-
pared to low consumption of carotene-containing
fruits and vegetables (for reviews see [16–19]). At
least one recent, very large meta-analysis [20] has
found signiﬁcant protective effects of increased lev-
els of dietary β-cryptoxanthin although recent trials
of beta-carotene and vitamin A supplements have
not shown any signiﬁcant reduction in lung cancer
risk; instead they showed an increased risk of lung
cancer death in the treated group [21–24]. Environ-
mental tobacco smoke (ETS, passive smoking) has
also been shown to be associated with increased risk
of lung cancer (for review see [3,4,25–27]) with a
recent prospective European study estimating that
between 16 and 24%of lung cancers in nonsmokers
and long-term ex-smokers were attributable to ETS
[28]. A recent meta-analysis of 22 studies showed
that exposure to workplace ETS increased risk of
lung cancer in workers by 24% and that this risk
was highly correlated with duration of exposure
[29]. These environmental risk factors cannot be
reviewed in detail here. There is little doubt that
the majority of lung cancer cases are attributable to
(i.e., would not occur in the absence of) cigarette
smoking and other behavioral and environmental
risk factors [2,7,25,30]. However, some investiga-
tors have long hypothesized that individuals differ
in their susceptibility to these environmental in-
sults (e.g. [31–34]). It is well known that muta-
tions and loss of heterozygosity at genetic loci such
as oncogenes and tumor suppressor genes are in-
volved in lung carcinogenesis (see [35,36] for re-
views) but most of these changes are thought to
be accumulated at the somatic cell level. However,
evidence has beenmounting that certain allelic vari-
ants at some genetic loci may affect susceptibil-
ity to lung cancer, although these effects may be
small. Furthermore, mounting epidemiologic evi-
dence has suggested lung cancer may show famil-
ial aggregation after adjusting for cigarette smok-
ing and other risk factors, and that differential sus-
ceptibility to lung cancer may be inherited in a
Mendelian fashion. There is evidence that both lung
cancer and smoking-associated cancer in general
have an inherited genetic component, but the exis-
tence of such a genetic component has not been def-
initely proven. This chapter will detail the evidence
suggesting the existence of inherited major sus-
ceptibility loci for lung cancer risk, and will relate
these risks to the well-known risks due to environ-
mental risk factors, particularly personal cigarette
smoking.

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2009-03-30T14:44:00.000-07:00

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studies. BMJ 2000; 321(7257):329.
25 Albert A, Samet J. Epidemiology of lung cancer. Chest
2003; 123(Suppl 1):21S–49S.
26 US Public Health Service. Surgeon General’s Advisory
Committee on Smoking and Health.Washington, DC: US
Public Health Service, 1964.
27 American Cancer Society. Cancer Facts and Figures. At-
lanta, GA: American Cancer Society, 2004.
28 Mackay J, Amos A. Women and tobacco. Respirology
2003; 8:123–30.
29 Siegfriend J. Woman and lung cancer: does estrogen
play a role? Lancet Oncol 2001; 2(8):606–13.
30 US Environmental Protection Agency. Respiratory
Health Effects of Passive Smoking: Lung Cancer and Other
Disorders.Washington, DC: US EPA, 1992. Report No.:
Publication EPA/600/6-90/006F.
31 USDHHS. The Health Consequences of Using Smokeless
Tobacco. A Report of the Advisory Committee to the Sur-
geon General. NIH Publication No 86-2874 1986 [cited
May 8, 2006]. Available from http://proﬁles.nlm.nih.
gov/NN/B/B/F/C/ /nnbbfc.pdf.

Smoking cessation interventions 2

2009-03-30T14:41:00.000-07:00

Sustained-release bupropion (Zyban)
Initially marketed as an atypical antidepressant,
sustained-release bupropion is hypothesized to pro-
mote smoking cessation by inhibiting the reuptake
of dopamine and norepinephrine in the central
nervous system [116] and acting as a nicotinic
acetylcholine receptor antagonist [117]. These neu-
rochemical effects are believed to modulate the
dopamine reward pathway and reduce the cravings
for nicotine and symptoms of withdrawal [112].
Because seizures are a dose-related toxicity
associated with bupropion, this medication is
contraindicated in patients with underlying seizure
disorders and in patients receiving concurrent ther-
apy with other forms of bupropion (Wellbutrin,
Wellbutrin SR, and Wellbutrin XL). Bupropion also
is contraindicated in patients with anorexia or bu-
limia nervosa and in patients who are undergo-
ing abrupt discontinuation of alcohol or sedatives
(including benzodiazepines) due to the increased
risk for seizures. The concurrent administration
of bupropion and a monoamine oxidase (MAO)
inhibitor is contraindicated and at least 14 days
should elapse between discontinuation of an MAO
inhibitor and initiation of treatment with bupro-
pion [118]. Although seizures were not reported in
the smoking cessation clinical trials, the incidence
of seizures with the sustained-release formulation
(Wellbutrin) used in the treatment of depression
was 0.1% among patients without a previous his-
tory of seizures [119]. For this reason, bupropion
should be used with extreme caution in patients
with a history of seizure, cranial trauma, patients
receiving medications known to lower the seizure
threshold, and patients with underlying severe hep-
atic cirrhosis. Bupropion is classiﬁed as a pregnancy
category C drug, meaning that either (a) animal
studies have demonstrated that the drug exerts ani-
mal teratogenic or embryocidal effects, but there are
no controlled studies inwomen, or (b) no studies are
available in either animals or women. Correspond-
ingly, themanufacturer recommends that this agent
be used during pregnancy only if clearly necessary
[118].Varenicline tartrate (Chantix)
The efﬁcacy of varenicline, a partial agonist selec-
tive for the a4b2 nicotinic acetylcholine receptor
[120,121), is believed to be the result of sustained,
low-level agonist activity at the receptor site com-
bined with competitive inhibition of nicotine bind-
ing. The partial agonist activity induces mod-
est receptor stimulation, which leads to increased
dopamine levels, thereby attenuating the symptoms
of nicotine withdrawal. In addition, by competi-
tively blocking the binding of nicotine to nicotinic
acetylcholine receptors in the central nervous sys-
tem, varenicline inhibits the surges of dopamine
release that occur following the inhalation of to-
bacco smoke. The latter effect might be effective in
preventing relapse by reducing the reinforcing and
rewarding effects of smoking [120]. The FDA classi-
ﬁes varenicline as a pregnancy category C drug, and
themanufacturer recommends that thismedication
be used during pregnancy only if the potential ben-
eﬁt justiﬁes the potential risk to the fetus [121].
Summary
Tobacco use remains prevalent among the popula-
tion and represents a matter of special public health
concern. It is the primary risk factor for the devel-
opment of lung cancer. It has been shown to cause
malignancies in other locations, aswell as numerous
other diseases. The body of knowledge of various
aspects of smoking behavior has largely increased
over the past two decades. Studies of factors predis-
posing to smoking initiation among youthmay pro-
vide important clues for the development of feasi-
ble and effective smoking prevention activities. The
knowledge of biobehavioral factors leading to devel-
opment of nicotine dependence may assist in pro-
vidingmore effective treatments to patientswho use
tobacco products. The ﬁve A’s approach (Ask about
tobacco use,Advise patients to quit,Assess readiness
to quit, Assist with quitting, and Arrange follow-
up) is described in the US Public Health Service
Clinical Practice Guideline for Treating Tobacco Use and
Dependence. Health care providers are encouraged to
implement at least brief interventions at each en-
counter with a patient who uses tobacco.

Nicotine replacement therapy

2009-03-30T14:40:00.000-07:00

In clinical trials, patients who use NRT products are
1.77 times as likely to quit smoking than are those
who receive placebo [7]. The main mechanism of
action of NRT products is thought to be a stimula-
tion of nicotine receptors in the ventral tegmental
area of the brain, which results in dopamine release
in the nucleus accumbens. The use of NRT is to re-
duce the physical withdrawal symptoms and to al-
leviate the physiologic symptoms of withdrawal, so
the smoker can focus on the behavioral and psy-
chological aspects of quitting before fully abstaining
nicotine. Key advantages of NRT are that patients
are not exposed to the carcinogens and other toxic
compounds found in tobacco and tobacco smoke,
and NRT provides slower onset of action than nico-
tine delivered via cigarettes, thereby eliminating the
near-immediate reinforcing effects of nicotine ob-
tained through smoking (Figure 1.3). NRT products
are pregnant if these patients are under medical su-
pervision [112]. Patients with temporomandibular
joint disease should not use the nicotine gum, and
patients smoking fewer than 10 cigarettes daily
should initiate NRT with caution and generally at
reduced dosages [112]. The safety and efﬁcacy of
NRT have not been established in adolescents, and
currently none of the NRT products are indicated
for use in this population [112,115].

Pharmaceutical aids for smoking cessation

2009-03-30T14:39:00.000-07:00

According to the Clinical Practice Guideline [112],
all patients attempting to quit should be encour-
aged to use one or more effective pharmacother-
apy agents for cessation except in the presence of
special circumstances. These recommendations are
supported by the results ofmore than 100 controlled
trials demonstrating that patients receiving pharma-
cotherapy are approximately twice as likely to re-
main abstinent long-term(greater than 5mo)when
compared to patients receiving placebo (Figure 1.2).
Although one would argue that pharmacotherapy
is costly and might not be a necessary component
of a treatment plan for each patient, it is the most
effective known method for maximizing the odds
of success for any given quit attempt, particularly
when combined with behavioral counseling [112].
Currently, seven marketed agents have an FDA-
approved indication for smoking cessation in the
US: ﬁve nicotine replacement therapy (NRT) for-
mulations (nicotine gum, nicotine lozenge, trans-
dermal nicotine patches, nicotine nasal spray, and
nicotine oral inhaler), sustained-release bupropion,
and varenicline tartrate. These are described in brief
below, and summaries of the prescribing informa-
tion for each medication are provided in Table 1.4.

Behavioral counseling

2009-03-30T14:37:00.000-07:00

Behavioral interventions play an integral role in
smoking cessation treatment, either alone or in con-
junction with pharmacotherapy. These interven-
tions, which include a variety of methods ranging
from self-help materials to individual cognitive–
behavioral therapy, enable individuals to more ef-
fectively recognize high-risk smoking situations, de-
velop alternative coping strategies, manage stress,
improve problem-solving skills, and increase social
support [113]. The Clinical Practice Guideline out-
lines a ﬁve-step framework that clinicians can apply
when assisting patients with quitting. Health care
providers should: (a) systematically identify all to-
bacco users, (b) strongly advise all tobacco users to
quit, (c) assess readiness to make a quit attempt, (d)
assist patients in quitting, and (e) arrange follow-up
contact. The steps have been described as the 5 A’s:
Ask, Advise, Assess, Assist, and Arrange follow-up
(Table 1.3). Due to the possibility of relapse, health
care providers should also provide patients with
brief relapse prevention treatment. Relapse preven-
tion reinforces the patient’s decision to quit, reviews
the beneﬁts of quitting, and assists the patient in re-
solving any problems arising from quitting [112].
The outlined strategy has been termed the 5 R’s
(Table 1.3): Relevance, Risks, Rewards, Roadblocks,
and Repetition. In the absence of time or expertise
for providingmore comprehensive counseling, clin-
icians are advised to (at a minimum), ask about to-
bacco use, advise tobacco users to quit, and refer
these patients to other resources for quitting, such
as a toll-free tobacco cessation quitline (1-800-QUIT
NOW, in the US).

Smoking cessation interventions

2009-03-30T14:36:00.000-07:00

Effective and timely administration of smoking ces-
sation interventions can signiﬁcantly reduce the risk
of smoking-related disease [110]. Recognizing the
complexity of tobacco use is a necessary ﬁrst step
in developing effective interventions and trials for
cessation and prevention. The biobehavioral model
of nicotine addiction and tobacco-related cancers
presents the complex interplay of social, psycho-
logical, and biological factors that inﬂuence tobacco
use and addiction (Figure 1.1). These factors in turn
mediate dependence, cessation, and relapse in most
individuals, and treatment has been developed to
addressmany of the factors noted in themodel [38].
The health care provider’s role
and responsibility
Health care providers are uniquely positioned to
assist patients with quitting, having both access to
quitting aids and commanding a level of respect that
renders themparticularly inﬂuential in advising pa-
tients on health-related issues. To date, physicians
have received the greatest attention in the scien-
tiﬁc community as providers of tobacco cessation
treatment. Although less attention has been paid to
other health care providers such as pharmacists and
nurses, they too are in a unique position to serve
the public and situated to initiate behavior change
among patients or complement the efforts of other
providers [64,111].
Fiore and associates conducted a meta-analysis
of 29 investigations in which they estimated that
compared with smokers who do not receive an in-
tervention from a clinician, patients who receive
a tobacco cessation intervention from a physician
clinician or a nonphysician clinician are 2.2 and
1.7 times as likely to quit smoking at 5 or more
months postcessation, respectively [112]. Although
brief advice from a clinician has been shown to
lead to increased likelihood of quitting, more in-
tensive counseling leads to more dramatic increases
in quit rates [112]. Because the use of pharma-
cotherapy agents approximately doubles the odds
of quitting [7,112], smoking cessation interventions
should consider combining pharmacotherapy with
behavioral counseling.
To assist clinicians and other health care providers
in providing cessation treatment, the US Public
Health Service has produced a Clinical Practice Guide-
line for the Treatment of Tobacco Use and Dependence
[112]. The Guideline is based on a systematic re-
viewand analysis of scientiﬁc literaturewhich yields
a series of recommendations and strategies to as-
sist health care providers in delivering smoking
cessation treatment. The Guideline emphasizes the
importance of systematic identiﬁcation of tobacco
users by health care workers and offering at least
brief treatment interventions to every patient who
uses tobacco. Among the most effective approaches
for quitting are behavioral counseling and pharma-
cotherapy, used alone or, preferably, in combination
[112].

Beneﬁts of quitting

2009-03-30T14:35:00.000-07:00

The reports of the US Surgeon General on the
health consequences of smoking, released in 1990
and 2004, summarize abundant and signiﬁcant
health beneﬁts associated with giving up tobacco
[9,104]. Beneﬁts noticed shortly after quitting (e.g.,
within 2weeks to 3months), include improvements
in pulmonary function and circulation. Within
1–9 months of quitting, the ciliary function of
the lung epithelium is restored. Initially, patients
might experience increased coughing as the lungs
clear excess mucus and tobacco smoke particu-
lates. In several months, smoking cessation results
inmeasurable improvements of lung function. Over
time, patients experience decreased coughing, sinus
congestion, fatigue, shortness of breath, and risk for
pulmonary infection and 1 year postcessation, the
excess risk for coronary heart disease is reduced to
half that of continuing smokers. After 5–15 years,
the risk for stroke is reduced to a rate similar to
that of people who are lifetime nonsmokers, and
10 years after quitting, an individual’s chance of
dying of lung cancer is approximately half that of
continuing smokers. Additionally, the risk of devel-
oping mouth, larynx, pharynx, esophagus, bladder,
kidney, or pancreatic cancer is decreased. Finally,
15 years after quitting, a risk for coronary heart dis-
ease is reduced to a rate similar of that of peoplewho
have never smoked. Smoking cessation can also lead
to a signiﬁcant reduction in the cumulative risk for
death from lung cancer, for males and females.
Smokers who are able to quit by age 35 can be
expected to live an additional 6–9 years compared
to those who continue to smoke [105]. Ossip-Klein
et al. [106] recently named tobacco use a “geriatric
health issue.” Indeed, a considerable proportion of
tobacco users continue to smoke well into their 70s
and 80s, despite the widespread knowledge of the
tobacco health hazards. Elderly smokers frequently
claim that the “damage is done,” and it is “too late
to quit;” however, a considerable body of evidence
refutes these statements. Even individuals who
postpone quitting until age 65 can incur up to four
additional years of life, compared with those who
continued to smoke [24,106]. Therefore, elderly
smokers should not be ignored as a potential target
for cessation efforts. Health care providers ought to
remember that it is never too late to advise their
elderly patients to quit and to incur health beneﬁts.
A growing body of evidence indicates that con-
tinued smoking after a diagnosis of cancer has
substantial adverse effects. For example, these
studies indicate that smoking reduces the over-
all effectiveness of treatment, while causing com-
plications with healing as well as exacerbating
treatment side effects, increases risk of developing
second primary malignancy, and decreases over-
all survival rates [36–38,107–109]. On the other
hand, the medical, health, and psychosocial bene-
ﬁts of smoking cessation among cancer patients are
promising. Gritz et al. [37] indicated that stopping
smoking prior to diagnosis and treatment can have a
positive inﬂuence on survival rates. Althoughmany
smoking cessation interventions are aimed at pri-
mary prevention of cancer, these results indicate
that there can be substantial medical beneﬁts for
individuals who quit smoking after they are diag-
nosed with cancer.

Genetics of tobacco use and dependence

2009-03-30T14:33:00.000-07:00

As early as 1958, Fisher hypothesized that the link
between smoking and lung cancer could be ex-
plained at least in part by shared genes that predis-
pose individuals to begin smoking as young adults
and to develop lung cancer later in adulthood [88].
More recently, tobacco researchers have begun to
explorewhether genetic factors do in fact contribute
toward tobacco use and dependence.
Tobacco use and dependence are hypothesized to
result from an interplay of many factors (includ-
ing pharmacologic, environmental and physiologic)
[77]. Some of these factors are shared within fam-
ilies, either environmentally or genetically. Studies
of families consistently demonstrate that, compared
to family members of nonsmokers, family members
of smokers aremore likely to be smokers also. How-
ever, in addition to shared genetic predispositions,
it is important to consider environmental factors
that promote tobacco use—siblings within the same
family share many of the same environmental in-
ﬂuences as well as the same genes. To differentiate
the genetic fromthe environmental inﬂuences, epi-
demiologists use adoption, twin, twins reared apart,
and linkage study designs [89].
Key to the adoption studies is the assumption that
if a genetic link for tobacco use exists, then tobacco
use behaviors (e.g., smoking status, number of years
smoked, number of cigarettes smoked per day) will
be more similar for persons who are related geneti-
cally (i.e., biologically) than for persons who are not
related genetically. Hence, one would expect to ob-
serve greater similarities between children and their
biological parents and siblings than would be ob-
served between children and their adoptive parents
or adopted siblings. Indeed, research has demon-
strated stronger associations (i.e., higher correlation
coefﬁcients) between biologically-related individu-
als, compared to nonbiologically-related individu-
als, for the reported number of cigarettes consumed
[90]. In recent years, it has become more difﬁcult
to conduct adoption studies, because of the reduced
number of intranational children available for adop-
tion [91]. Additionally, delayed adoption (i.e., time
elapsed between birth and entry into the new fam-
ily) is common with international adoptions and
might lead to an overestimation of genetic effects
if early environmental inﬂuences are attributed to
genetic inﬂuences [92].
In twin studies, identical (monozygotic) twins
and fraternal (dizygotic) twins are compared. Iden-
tical twins share the same genes; fraternal twins,
like ordinary siblings, share approximately 50% of
their genes. If a genetic link exists for the phe-
nomenon under study, then one would expect to
see a greater concordance in identical twins than
in fraternal twins. Thus, in the case of tobacco
use, one would expect to see a greater proportion
of identical twins with the same tobacco use be-
havior than would be seen with fraternal twins.
Statistically, twin studies aim to estimate the per-
centage of the variance in the behavior that is
due to (1) genes (referred to as the “heritability”),
(2) shared (within the family) environmental ex-
periences, and (3) nonshared (external from the
family) environmental experiences [91]. A num-
ber of twin studies of tobacco use have been con-
ducted in recent years. These studies have largely
supported a genetic role [91,93]; higher concor-
dance of tobacco use behavior is evident in identical
twins than in fraternal twins. The estimated aver-
age heritability for smoking is 0.53 (range, 0.28–
0.84) [93,94]; approximately half of the variance
in smoking appears to be attributable to genetic
factors.
Recent advances in the mapping of the human
genome have enabled researchers to search for
genes associated with speciﬁc disorders, including
tobacco use. Using a statistical technique called link-
age analysis, it is possible to identify genes that pre-
dict a trait or disorder. This process is not based on
prior knowledge of a gene’s function, but rather it
is determined by examining whether the trait or
disorder is coinherited with markers found in spec-
iﬁed chromosomal regions. Typically, these types
of investigations involve collection of large family
pedigrees, which are studied to determine inheri-
tance of the trait or disorder. This method works
well when a single gene is responsible for the out-
come; however, it becomes more difﬁcult when
multiple genes have an impact, such as with to-
bacco use. In linkage studies of smoking, it is com-
mon for investigators to identify families, ideally
with two or more biologically-related relatives that
have the trait or disorder under study (referred to
as affected individuals, in this case, smokers) and
other unaffected relatives. For example, data from
affected sibling pairs with parents is a common de-
sign in linkage analysis. A tissue sample (typically
blood) is taken fromeach individual, and the sample
undergoes genotyping to obtain information about
the study participant’s unique genetic code. If a
gene in a speciﬁc region of a chromosome is as-
sociated with smoking, and if a genetic marker is
linked (i.e., in proximity), then the affected pairs
(such as affected sibling pairs) will have increased
odds for sharing the same paternal/maternal gene
[91].
As genetic research moves forward, new clues
provide insight into which genes might be promis-
ing “candidates” as contributors to tobacco use and
dependence. Currently, there are two general lines
of research related to candidate genes for smoking.
One examines genes that affect nicotine pharmaco-
dynamics (the way that nicotine affects the body)
and the other examines genes that affect nicotine
pharmacokinetics (the way that the body affects
nicotine). A long list of candidate genes are being
examined—some of the most extensively explored
involve (a) the dopamine reward pathway (e.g.,
those related to dopamine synthesis, receptor acti-
vation, reuptake, and metabolism) and (b) nicotine
metabolism via the cytochrome P450 liver enzymes
(speciﬁcally, CYP2A6 and CYP2D6).
In summary, each of these types of study designs
supports the hypothesis that genetics inﬂuence the
risk for a wide range of tobacco-related phenotypes,
such as ever smoking, age at smoking onset, level
of smoking, ability to quit, and the metabolic path-
ways of nicotine (e.g., see [45,89,95–99]). But given
that there are many predictors of tobacco use and
dependence, of which genetic predisposition is just
one piece of a complex puzzle, it is unlikely that so-
ciety will move toward widespread genotyping for
early identiﬁcation of individuals who are at risk
for tobacco use. Perhaps a more likely use of ge-
netics as related to tobacco use is its potential for
improving our treatment for dependence [91]. If
genetic research leads to new knowledge regarding
the mechanisms underlying the development and
maintenance of dependence, it is possible that new,
more effective medications might be created. Fur-
thermore, through pharmacogenomics research we
might gain improved knowledge as to which pa-
tients, based on their genetic proﬁles, would be best
treatedwithwhichmedications. Researchers are be-
ginning to examine how DNA variants affect health
outcome with pharmacologic treatments, with a
goal of determining which genetic proﬁles respond
most favorably to speciﬁc pharmaceutical aids for
cessation (e.g. [98,100–103]).

Nicotine addiction

2009-03-30T14:32:00.001-07:00

Nicotine has come to be regarded as a highly addic-
tive substance. Judging by the current diagnostic cri-
teria, tobacco dependence appears to be quite preva-
lent among cigarette smokers; more than 90% of
smokers meet the DSM-IV (Diagnostic and Statisti-
calManual ofMental Disorders) criteria for nicotine
dependence [76]. Research has shown that nico-
tine acts on the brain to produce a number of ef-
fects [77,78] and immediately after exposure, nico-
tine induces a wide range of central nervous sys-
tem, cardiovascular, and metabolic effects. Nicotine
stimulates the release of neurotransmitters, in-
ducing pharmacologic effects, such as pleasure
and reward (dopamine), arousal (acetylcholine,
norepinephrine), cognitive enhancement (acetyl-
choline), appetite suppression (norepinephrine),
learning and memory enhancement (glutamate),
mood modulation and appetite suppression (sero-
tonin), and reduction of anxiety and tension
(β-endorphin and GABA) [78]. Upon entering the
brain, a bolus of nicotine activates the dopamine re-
ward pathway, a network of nervous tissue in the
brain that elicits feelings of pleasure and stimulates
the release of dopamine.
Although withdrawal symptoms are not the only
consequence of abstinence, most cigarette smok-
ers do experience craving and withdrawal on ces-
sation [79], and, therefore, relapse is common [80].
The calming effect of nicotine reported by many
users is usually associated with a decline in with-
drawal effects rather than direct effects on nicotine
[53]. This rapid dose-response, along with the short
half-life of nicotine (t1 / 2 = 2 h), underlies tobacco
users’ frequent, repeated administration, thereby
perpetuating tobacco use and dependence. Tobacco
users become proﬁcient in titrating their nicotine
levels throughout the day to avoid withdrawal
symptoms, to maintain pleasure and arousal, and
to modulate mood. Withdrawal symptoms include
depression, insomnia, irritability/frustration/anger,
anxiety, difﬁculty concentrating, restlessness, in-
creased appetite/weight gain, and decreased heart
rate [81,82].
The assumption that heavy daily use (i.e., 15–
30 cigarettes per day), is necessary for dependence
to develop is derived from observations of “chip-
pers,” adult smokers who have not developed de-
pendence despite smoking up to ﬁve cigarettes per
day for many years [83,84]. Chippers do not tend
to differ from other smokers in their absorption and
metabolism of nicotine, causing some investigators
to suggest that this level of consumptionmay be too
low to cause nicotine dependence. However, these
atypical smokers are usually eliminated from most
studies, which are routinely limited to smokers of
at least 10 cigarettes per day [83].
Signs of dependence on nicotine have been re-
ported among adolescent smokers, with approx-
imately one ﬁfth of them exhibiting adult-like
dependence [85]. Although, lengthy and regular
tobacco use has been considered necessary for
nicotine dependence to develop [68], recent re-
ports have raised concerns that nicotine depen-
dence symptoms can develop soon after initiation,
and that these symptoms might lead to smoking
intensiﬁcation [79,86]. Adolescent smokers, who
use tobacco regularly, tend to exhibit high craving
for cigarettes and substantial levels of withdrawal
symptoms [87].

Factors explaining tobacco use

2009-03-30T14:30:00.000-07:00

Smoking initiation
In the United States, smoking initiation typically
occurs during adolescence. About 90% of adult
smokers have tried their ﬁrst cigarette by 18 years
of age and 70% of daily smokers have become
regular smokers by that age [67,68]. Because most
adolescents who smoke at least monthly continue
to smoke into adulthood, youth-oriented tobacco
preventions and cessation strategies are warranted
[67,68]. Since the mid-1990s, by 2004, the past-
month prevalence had decreased by 56% in 8th
graders, 47% in 10th graders, and 32% in 12th
graders [69]. In recent years, however, this down-
ward trend has decelerated [69]. The downward
trend is unlikely to be sustained without steady and
systematic efforts by health care providers in pre-
venting initiation of tobacco use and assisting young
smokers in quitting.
A wide range of sociodemographic, behavioral,
personal, and environmental factors have been ex-
amined as potential predictors of tobacco exper-
imentation and initiation of regular tobacco use
among adolescents. For example, it has been sug-
gested that the prevalence of adolescent smok-
ing is related inversely to parental socioeconomic
status and adolescent academic performance [68].
Other identiﬁed predictors of adolescent smoking
include social inﬂuence and normative beliefs, neg-
ative affect, outcome expectations associated with
smoking, resistance skills (self-efﬁcacy), engaging in
other risk-taking behaviors, exposure to smoking in
movies, and having friends who smoke [70–75].
Although numerous studies have been successful
in identifying predictors of smoking initiation, few
studies have identiﬁed successful methods for pro-
moting cessation among youth, despite the ﬁnding
that in 2005,more than half of high school cigarette
smokers have tried to quit smoking in the past year
and failed [52]. These results conﬁrm the highly
addictive nature of tobacco emphasizing the need
for more effective methods for facilitating cessation
among the young.

Smokeless tobacco

2009-03-30T14:29:00.000-07:00

Smokeless tobacco products, also commonly called
“spit tobacco,” are placed in the mouth to allow ab-
sorption of nicotine through the buccalmucosa. Spit
tobacco includes chewing tobacco and snuff. Chew-
ing tobacco,which is typically available in loose leaf,
plug, and twist formulations, is chewed or parked in
the cheek or lower lip. Snuff, commonly available as
loose particles or sachets (resembling tea bags), has
a much ﬁner consistency and is generally held in
the mouth and not chewed. Most snuff products
in the United States are classiﬁed as moist snuff.
The users park a “pinch” (small amount) of snuff
between the cheek and gum (also known as dip-
ping) for 30 minutes or longer. Dry snuff is typically
sniffed or inhaled through the nostrils; it is used less
commonly [64].
In 2004, an estimated 3.0%ofAmericans 12 years
of age and older had used spit tobacco in the past
month. Men used it at higher rates (5.8%) than
women (0.3%) [60]. The prevalence of spit tobacco
is the highest among 18- to 25-year-olds and is sub-
stantially higher among American Indians, Alaska
natives, residents of the southern states, and ru-
ral residents [61,66]. The consumption of chew-
ing tobacco has been declining since themid-1980s;
conversely, in 2005, snuff consumption increased by
approximately 2%over the previous year [66], pos-
sibly because tobacco users are consuming snuff in-
stead of cigarettes in locations and situations where
smoking is banned.

Forms of tobacco

2009-03-30T14:28:00.000-07:00

Smoked tobacco
Cigarettes have been the most widely used form of
tobacco in theUnited States for several decades [51],
yet in recent years, cigarette smoking has been de-
clining steadily among most population subgroups.
In 2005, just over half of ever smokers reported be-
ing former smokers [3]. However, a considerable
proportion of the population continues to smoke.
In 2005, an estimated 45.1 million adult Americans
(20.9%) were current smokers; of these, 80.8% re-
ported to smoking every day, and 19.2% reported
smoking some days [7]. The prevalence of smoking
varies considerably across populations (Table 1.2),
with a greater proportion of men (23.9%) than
women (18.1%) reporting current smoking. Per-
sons of Asian or Hispanic origin exhibit the low-
est prevalence of smoking (13.3 and 16.2%, respec-
tively), and American Indian/Alaska natives exhibit
the highest prevalence (32.0%). Also, the preva-
lence of smoking among adults varies widely across
the United States, ranging from 11.5% in Utah to
28.7% in Kentucky [51]. Twenty-three percent of
high school students report current smoking, and
among boys, 13.6% report current use of smoke-
less tobacco, and 19.2%currently smoke cigars [52].
These ﬁgures are of particular concern, because
nearly 90% of smokers begin smoking before the
age of 18 years [53].
Other common forms of burned tobacco in the
United States include cigars, pipe tobacco, and bidis.
Cigars represent a roll of tobacco wrapped in leaf to-
bacco or in any substance containing tobacco [54].
Cigars’ popularity has somewhat increased over the
past decade [55]. The latter phenomenon is likely
to be explained by a certain proportion of smok-
ers switching cigarettes for cigars and by adoles-
cents’ experimentation with cigars [56]. In 1998,
approximately 5%of adults had smoked at least one
cigar in the past month [57]. The nicotine content
of cigars sold in the United States ranged from 5.9
to 335.2 mg per cigar [58] while cigarettes have a
narrow range of total nicotine content, between 7.2
and 13.4 mg per cigarette [59]. Therefore, one large
cigar, which could contain as much tobacco as an
entire pack of cigarettes is able to deliver enough
nicotine to establish and maintain physical depen-
dence [59].
Pipe smoking has been declining steadily over the
past 50 years [60]. It is a form of tobacco use seen
among less than 1% of Americans [60]. Bidi smok-
ing is a more recent phenomenon in the United
States. Bidis are hand-rolled brown cigarettes im-
portedmostly fromSoutheast Asian countries. Bidis
arewrapped in a tendu or temburni leaf [61]. Visually,
they somewhat resemble marijuana joints, which
might make them attractive to certain groups of
the populations. Bidis are available in multiple ﬂa-
vors (e.g., chocolate, vanilla, cinnamon, strawberry,
cherry, mango, etc.), which might make them par-
ticularly attractive to younger smokers. A survey
of nearly 64,000 people in 15 states in the United
States revealed that young people (18–24 years of
age) reported higher rates of ever (16.5%) and
current (1.4%) use of bidis then among older adults
(ages 25 plus years). With respect to sociodemo-
graphic characteristics, the use of bidis is most com-
mon among males, African Americans, and con-
comitant cigarette smokers [62].Although featuring
less tobacco than standard cigarettes, bidis expose
their smokers to considerable amounts of hazardous
compounds. A smoking machine-based investiga-
tion found that bidis deliver three times the amount
of carbon monoxide and nicotine and almost ﬁve
times the amount of tar found in conventional
cigarettes [63].

Percentage of current cigarette smokersa aged ≥18 years

2009-03-30T14:27:00.001-07:00

Table 1.2 Percentage of current cigarette smokersa aged ≥18 years, by selected characteristics—National Health
Interview Survey, United States, 2005.
Characteristic Category Men (n = 13,762) Women (n = 17,666) Total (n = 31,428)
Race/ethnicityb White, non-Hispanic 24.0 20.0 21.9
Black, non-Hispanic 26.7 17.3 21.5
Hispanic 21.1 11.1 16.2
American Indian/Alaska Native 37.5 26.8 32.0
Asianc
20.6 6.1 13.3
Educationd 0–12 years (no diploma) 29.5 21.9 25.5
GEDe (diploma) 47.5 38.8 43.2
High school graduate 28.8 20.7 24.6
Associate degree 26.1 17.1 20.9
Some college (no degree) 26.2 19.5 22.5
Undergraduate degree 11.9 9.6 10.7
Graduate degree 6.9 7.4 7.1
Age group (yrs) 18–24 28.0 20.7 24.4
25–44 26.8 21.4 24.1
45–64 25.2 18.8 21.9
≥65 8.9 8.3 8.6
Poverty level
f
At or above 23.7 17.6 20.6
Below 34.3 26.9 29.9
Unknown 21.2 16.1 18.4
Total 23.9 18.1 20.9
aPersons who reported having smoked at least 100 cigarettes during their lifetime and at the time of the interview reported
smoking every day or some days; excludes 296 respondents whose smoking status was unknown.
bExcludes 314 respondents of unknown or multiple racial/ethnic categories or whose racial/ethnic category was unknown.
c
Excludes Native Hawaiians or other Paciﬁc Islanders.
dPersons aged ≥25 years, excluding 339 persons with unknown level of education.
eGeneral Educational Development.
f
Calculated on the basis of US Census Bureau 2004 poverty thresholds.
Source: Reference [7].

Smoking among lung cancer patients

2009-03-30T14:25:00.000-07:00

Tobacco use among patients with cancer is a se-
rious health problem with signiﬁcant implications
for morbidity and mortality [36–39]. Evidence in-
dicates that continued smoking after a diagnosis
with cancer has substantial adverse effects on treat-
ment effectiveness [40], overall survival [41], risk
of second primary malignancy [42], and increases
the rate and severity of treatment-related complica-
tions such as pulmonary and circulatory problems,
infections, impaired would healing, mucositis, and
Xerostomia [43,44].
Despite the strong evidence for the role of smok-
ing in the development of cancer, many cancer pa-
tients continue to smoke. Speciﬁcally, about one
third of cancer patients who smoked prior to their
diagnoses continue to smoke [45] and among pa-
tients received surgical treatment of stage I nonsmall
cell lung cancer [46] found only 40%who were ab-
stinent 2 years after surgery. Davison and Duffy [47]
reported that 48% of former smokers had resumed
regular smoking after surgical treatment of lung
cancer. Therefore, among patients with smoking-
related malignancies, the likelihood of a positive
smoking history at and after diagnosis is high.
Patients who are diagnosed with lung cancermay
face tremendous challenges and motivation to quit
after a cancer diagnosis can be inﬂuenced by a range
of psychological variables. Schnoll and colleagues
[48] reported that continued smoking among pa-
tients with head and neck and lung cancer is asso-
ciated with lesser readiness to quit, having relatives
who smoke at home, greater time between diag-
noses and assessment, greater nicotine dependence,
lower self-efﬁcacy, lower risk perception, fewer per-
ceived pros and greater cons to quitting, more fa-
talistic beliefs, and higher emotional distress. Lung
cancer patients should be advised to quit smoking,
but once they are diagnosed, some might feel that
there is nothing to be gained from quitting [49].
Smoking cessation should be a matter of special
concern throughout cancer diagnosis, treatment,
and the survival continuum, and the diagnosis of
cancer should be used as a “teachable moment”
to encourage smoking cessation among patients,
family members, and signiﬁcant others [37]. The

Second-hand smoke and lung cancer

2009-03-30T14:24:00.000-07:00

While active smoking has been shown to be the
main preventable cause of lung cancer, second-
hand smoke contains the same carcinogens that are
inhaled by smokers [30]. Consequently, there has
been a concern since release of the 1986 US Sur-
geon General’s report [31] concluding that second-
hand smoke causes cancer among nonsmokers and
smokers. Although estimates vary by exposure lo-
cation (e.g., workplace, car, home), the 2000 Na-
tional Household Interview Survey estimates that a
quarter of the US population is exposed to second-
hand smoke [32]. Second-hand smoke is the third
leading cause of preventable deaths in the United
States [33], and it has been estimated that expo-
sure to second-hand smoke kills more than 3000
adult nonsmokers from lung cancer [34]. Accord-
ing to Glantz and colleagues, for every eight smok-
ers who die froma smoking-attributable illness, one
additional nonsmoker dies because of second-hand
smoke exposure [35].
Since 1986, numerous additional studies have
been conducted and summarized in the 2006 US
Surgeon General’s report on “The Health Conse-
quences of Involuntary Exposure of Tobacco Smoke.” The
report’s conclusions based on this additional ev-
idence are consistent with the previous reports:
exposure to second-hand smoke increases risk of
lung cancer. More than 50 epidemiologic stud-
ies of nonsmokers’ cigarette smoke exposure at
the household and/or in the workplace showed
an increased risk of lung cancer associated with
second-hand smoke exposure [34]. This means that
20 years after second-hand smoke was ﬁrst es-
tablished as a cause of lung cancer in lifetime
nonsmokers, the evidence supporting smoking ces-
sation and reduction of second-hand smoke expo-
sure continues to mount. Eliminating second-hand
smoke exposure at home, in the workplaces, and
other public places appears to be essential for re-
ducing the risk of lung cancer development among
nonsmokers.

CHAPTER 1 Smoking Cessation 2

2009-03-30T14:23:00.000-07:00

annual number of deaths from breast, colon, and
prostate cancer combined [15]. Recent advances in
technology have enabled earlier diagnoses, and ad-
vances in surgery, radiation therapy, imaging, and
chemotherapy have produced improved responses
rates. However, despite these efforts, overall sur-
vival has not been appreciably affected in 30 years,
and only 12–15% of patients with lung cancer are
being cured with current treatment approaches
[16]. The prognosis of lung cancer depends largely
on early detection and immediate, premetastasis
stage treatment [17]. Prevention of lung cancer
is the most desirable and cost-efﬁcient approach
to eradicating this deadly condition. Numerous
epidemiologic studies consistently deﬁne smoking
as the major risk factor for lung cancer (e.g. [18–
20]). The causal role of cigarette smoking in lung
cancer mortality has been irrefutably established
in longitudinal studies, one of which lasted as long
as 50 years [21]. Tobacco smoke, which is inhaled
either directly or as second-hand smoke, contains
an estimated 4000 chemical compounds, including
over 60 substances that are known to cause cancer
[22]. Tobacco irritants and carcinogens damage the
cells in the lungs, and over time the damaged cells
may become cancerous. Cigarette smokers have
lower levels of lung function than nonsmokers
[9,23], and quitting smoking greatly reduces
cumulative risk for developing lung cancer [24].
The association of smokingwith the development
of lung cancer is the most thoroughly documented
causal relationship in biomedical history [25]. The
link was ﬁrst observed in the early 1950s through
the research of Sir Richard Doll, whose pioneering
research has, perhaps more so than any other epi-
demiologist of his time, altered the landscape of dis-
ease prevention and consequently saved millions of
lives worldwide. In two landmark US Surgeon Gen-
erals’ reports publishedwithin a 20-year interval (in
1964 [26] and in 2004 [9]), literature syntheses fur-
ther documented the strong link between smoking
and cancer. Compared to never smokers, smokers
have a 20-fold risk of developing lung cancer, and
more than 87% of lung cancers are attributable to
smoking [27]. The risk for developing lung cancer
increases with younger age at initiation of smoking,
greater number of cigarettes smoked, and greater
number of years smoked [11].Women smoking the
same amount as men experience twice the risk of
developing lung cancer [28,29].

CHAPTER 1 Smoking Cessation

2009-03-30T14:20:00.000-07:00

Overview
Tobacco use is a public health issue of enormous
importance, and smoking is the primary risk factor
for the development of lung cancer. Considerable
knowledge has been gained with respect to biobe-
havioral factors leading to smoking initiation and
development of nicotine dependence. Smoking ces-
sation provides extensive health beneﬁts for every-
one. State-of-the-art treatment for smoking cessa-
tion includes behavioral counseling in conjunction
with one or more FDA-approved pharmaceutical
aids for cessation. The US Public Health Service Clin-
ical Practice Guideline for Treating Tobacco Use and De-
pendence advocates a ﬁve-step approach to smoking
cessation (Ask about tobacco use, Advise patients to
quit, Assess readiness to quit, Assist with quitting,
and Arrange follow-up). Health care providers are
encouraged to provide at least brief interventions at
each encounter with a patient who uses tobacco.
Introduction
More than two decades ago, the former US Surgeon
General C. Everett Koop stated that cigarette smok-
ing is the “chief, single, avoidable cause of death in
our society and the most important public health
issue of our time” [1]. This statement remains true
today. In the United States, cigarette smoking is the
primary known cause of preventable deaths [2],
resulting in nearly 440,000 deaths each year [3].
The economic implications are enormous: more
than $75 billion in medical expenses and over $81
billion in loss of productivity as a result of pre-
mature death are attributed to smoking each year
[4–8].While the public often associates tobacco use
with elevated cancer risk, the negative health con-
sequences are much broader. The 2004 Surgeon
General’s Report on the health consequences of
smoking [9] provides compelling evidence of the ad-
verse impact of smoking and concluded that smok-
ing harms nearly every organ in the body (Table
1.1). In 2000, 8.6 million persons in the United
States were living with an estimated 12.7 mil-
lion smoking-attributable medical conditions [10].
There is convincing evidence that stopping smok-
ing is associated with immediate as well as long-
term health beneﬁts, including reduced cumulative
risk for cancer. This is true even in older individu-
als, and in patients who have been diagnosed with
cancer [11].
Smoking and lung cancer
In the United States, approximately 85% of all
lung cancers are in people who smoke or who
have smoked [3]. Lung cancer is fatal for most
patients. The estimated number of deaths of lung
cancer will exceed 1.3 million annually early in the
third millennium [12]. Lung cancer is the leading
cause of cancer-related deaths among Americans
of both genders, with 174,470 estimated newly
diagnosed cases and 162,460 deaths [13,14]. The
number of deaths due to lung cancer exceeds the
Table 1.1 Health consequences of smoking.
Cancer Acute myeloid leukemia
Bladder
Cervical
Esophageal
Gastric
Kidney
Laryngeal
Lung
Oral cavity and pharyngeal
Pancreatic
Cardiovascular
diseases
Abdominal aortic aneurysm
Coronary heart disease (angina pectoris,
ischemic heart disease, myocardial
infarction, sudden death)
Cerebrovascular disease (transient
ischemic attacks, stroke)
Peripheral arterial disease
Pulmonary Acute respiratory illnesses
diseases Pneumonia
Chronic respiratory illnesses
Chronic obstructive pulmonary
disease
Respiratory symptoms (cough,
phlegm, wheezing, dyspnea)
Poor asthma control
Reduced lung function in infants
exposed (in utero) to maternal
smoking
Reproductive
effects
Reduced fertility in women
Pregnancy and pregnancy outcomes
Premature rupture of membranes
Placenta previa
Placental abruption
Preterm delivery
Low infant birth weight
Infant mortality (sudden infant death
syndrome)
Other
effects
Cataract
Osteoporosis (reduced bone density in
postmenopausal women, increased risk
of hip fracture)
Periodontitis
Peptic ulcer disease (in patients who are
infected with Helicobacter pylori)
Surgical outcomes
Poor wound healing
Respiratory complications

Role of smoking in lung cancer

2009-03-27T06:23:00.000-07:00

Lung cancer is the most common cause of cancer
death in both men and women. High incidence of lung cancer over the past half century is directly linked to smoking.
Lung cancer

Smoking is responsible for 90% of all cancer deaths
Cases of lung cancer is increasing. Lung cancer is responsible for more deaths from cancer of colon and rectal cancer, breast cancer and prostate cancer combined.
Lung cancer is mainly that people over 45 years. At the time of each set of symptoms, the spread is usually the case.

Smoking

Lung cancer is directly related to smoking. More than 40 carcinogens have been identified in cigarette smoke. The risk of lung cancer is directly related to the number of cigarettes. Change in consumption of high-tar filter cigarettes Mufltrp to low tar cigarettes, which corresponds to a change in squamous cell carcinoma to adenocarcinoma. A long-term smoking cessation, and eliminate the risk of lung cancer. Up to 40% in newly diagnosed lung cancer of former smokers. (Do not the average duration of 9 years